Compositions and methods for reducing spliceopathy and treating rna dominance disorders

ABSTRACT

The disclosure features compositions and methods for the treatment of disorders associated with improper ribonucleic acid (RNA) splicing, including disorders characterized by nuclear retention of RNA transcripts containing aberrantly expanded repeat regions that bind and sequester splicing factor proteins. Disclosed herein are interfering RNA constructs that suppress the expression of RNA transcripts containing expanded repeat regions, as well as viral vectors, such as adeno-associated viral vectors, encoding such interfering RNA molecules. For example, the disclosure features interfering RNA molecules, such as siRNA, miRNA, and shRNA constructs, that anneal to dystrophia myotonica protein kinase (DMPK) RNA transcripts and attenuate the expression of DMPK RNA containing expanded CUG trinucleotide repeats. Using the compositions and methods described herein, a patient having an RNA dominance disorder, such as a human patient having myotonic dystrophy, among other conditions described herein, may be administered an interfering RNA construct or vector containing the same so as to reduce the occurrence of spliceopathy in the patient, thereby treating an underlying etiology of the disease.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under Grant No. R03AR056107, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to the field of nucleic acid biotechnology andprovides compositions and methods for treating genetic disordersassociated with improper ribonucleic acid splicing.

BACKGROUND OF THE INVENTION

The expression and nuclear retention of endogenous ribonucleic acid(RNA) transcripts containing aberrantly expanded repeat regions leads tothe onset of RNA dominance, a pathology that underlies various heritablegenetic disorders, including myotonic dystrophy type 1, among others.Myotonic dystrophy is the most common form of muscular dystrophy, andoccurs with an estimate frequency of about 1 in 7,500 human adults. RNAdominance results from a gain-of-function mutation in RNA transcriptsthat imparts these molecules with undesired biological activity. Inmyotonic dystrophy, RNA dominance is effectuated by the presence ofexpanded CUG trinucleotide repeats in RNA transcript that encodedystrophia myotonica protein kinase (DMPK), which sequester RNA proteinsthat control RNA splicing, such as muscleblind-like protein, by virtueof the elevated avidity of these expansion regions for such splicingfactor proteins. There is a paucity of strategies available forsuccessfully treating and ameliorating the symptoms of myotonicdystrophy, among other disorders associated with RNA dominance, andthere remains a need for effective therapeutics for these diseases.

SUMMARY OF THE INVENTION

Described herein are compositions and methods useful for reducing theoccurrence of spliceopathy and for treating disorders associated withribonucleic acid (RNA) dominance, a pathology that is induced by theexpression and nuclear retention of messenger RNA (mRNA) transcriptscontaining expanded repeat regions that bind and sequester splicingfactor proteins, thereby interfering with the proper splicing of variousmRNA transcripts. The compositions described herein that may be used totreat such disorders include nucleic acids containing interfering RNAconstructs that suppress the expression of RNA transcripts containingaberrantly expanded repeat regions, such as siRNA, miRNA, and shRNAconstructs that anneal to portions of nuclear-retained, repeat-expandedRNA transcripts and promote the degradation of these pathologicaltranscripts by way of various cellular processes. The present disclosureadditionally features vectors, such as viral vectors, encoding suchinterfering RNA constructs. Exemplary viral vectors described hereinthat encode interfering RNA constructs (e.g., siRNA, miRNA, or shRNA)for suppressing the expression of RNA transcripts containing aberrantlyexpanded repeat regions are adeno-associated viral (AAV) vectors, suchas pseudotyped AAV2/8 and AAV2/9 vectors.

Using the compositions and methods described herein, a patientexperiencing a spliceopathy and/or having a disease associated with RNAdominance, such as myotonic dystrophy, among others, can be administereda nucleic acid containing an interfering RNA construct, or a vectorencoding the same, so as to reduce the expression of RNA transcriptscontaining expanded repeat regions and release splicing factor proteinsthat are sequestered by repeat-expanded RNA. For example, thecompositions and methods described herein can be used to treat patientshaving myotonic dystrophy, as such patients may be administered aninterfering RNA construct or a viral vector, such as an AAV vector,encoding such a construct, thereby reducing the expression of RNAtranscripts encoding dystrophia myotonica protein kinase (DMPK).Wild-type DMPK RNA constructs typically contain from about 5 to about 37CUG trinucleotide repeats in the 3′ untranslated region (UTR) of suchtranscripts. Patients having myotonic dystrophy, however, express DMPKRNA transcripts that contain 50 or more CUG repeats. The compositionsand methods described herein can be used to treat patients expressingthis mutant DMPK RNA, thereby releasing splicing factors to orchestratethe proper splicing of proteins associated with muscle function andtreating an underlying cause of myotonic dystrophy. Similarly, thecompositions and methods described herein can be used to reducespliceopathy in, and treat one or more underlying causes of, variousother disorders associated with RNA dominance and the expression ofrepeat-expanded RNA transcripts.

The present disclosure is based, in part, on the surprising discoverythat interfering RNA constructs that anneal to repeat-expanded RNAtargets at sites distal from the expanded repeat region can be used tosuppress the expression of such RNA transcripts and effectively releasesplicing factor proteins that would otherwise be sequestered by thesemolecules. The compositions and methods described herein can thusattenuate the expression and nuclear retention of pathological RNAtranscripts without the need to contain complementary nucleotide repeatmotifs. This property provides an important clinical benefit. Nucleotiderepeats are ubiquitous in mammalian genomes, such as in the genomes ofhuman patients. The use of interfering RNA constructs that do not havenucleotide repeats, but rather anneal to other regions of a target RNAtranscript, enables the selective suppression of transcripts that giverise to RNA dominance disorders without disrupting the expression ofother transcripts, such as those encoding other genes that happen tocontain nucleotide repeat regions but that do not aberrantly sequestersplicing factor proteins. Using the compositions and methods describedherein, the expression of RNA transcripts that contain pathologicalnucleotide repeat expansions can be diminished, while preserving theexpression of important healthy RNA transcripts (for example, an RNAtranscript encoding a non-target gene that happens to contain anucleotide repeat), as well as their downstream protein products.

In a first aspect, the invention features a viral vector containing oneor more transgenes encoding an interfering RNA. For example, the viralvector may contain from one to five such transgenes, from one to 10 suchtransgenes, from one to 15 such transgenes, from one to 20 suchtransgenes, from one to 50 such transgenes, from one to 100 suchtransgenes, from one to 1,000 such transgenes, or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1,000, or more, suchtransgenes). The interfering RNA(s) may be at least 5, at least 10, atleast 17, at least 19, or more, nucleotides in length, (e.g., at least5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or more,nucleotides in length, such as from 17 to 24, 18 to 23, or 19 to 22nucleotides in length).

The interfering RNA(s) may, e.g., each be, independently, from 10-35nucleotides in length. In some embodiments, the interfering RNA(s) are10 nucleotides in length. In some embodiments, the interfering RNA(s)are 11 nucleotides in length. In some embodiments, the interferingRNA(s) are 12 nucleotides in length. In some embodiments, theinterfering RNA(s) are 13 nucleotides in length. In some embodiments,the interfering RNA(s) are 14 nucleotides in length. In someembodiments, the interfering RNA(s) are 15 nucleotides in length. Insome embodiments, the interfering RNA(s) are 16 nucleotides in length.In some embodiments, the interfering RNA(s) are 17 nucleotides inlength. In some embodiments, the interfering RNA(s) are 18 nucleotidesin length. In some embodiments, the interfering RNA(s) are 19nucleotides in length. In some embodiments, the interfering RNA(s) are20 nucleotides in length. In some embodiments, the interfering RNA(s)are 21 nucleotides in length. In some embodiments, the interferingRNA(s) are 22 nucleotides in length. In some embodiments, theinterfering RNA(s) are 23 nucleotides in length. In some embodiments,the interfering RNA(s) are 24 nucleotides in length. In someembodiments, the interfering RNA(s) are 25 nucleotides in length. Insome embodiments, the interfering RNA(s) are 26 nucleotides in length.In some embodiments, the interfering RNA(s) are 27 nucleotides inlength. In some embodiments, the interfering RNA(s) are 28 nucleotidesin length. In some embodiments, the interfering RNA(s) are 29nucleotides in length. In some embodiments, the interfering RNA(s) are30 nucleotides in length. In some embodiments, the interfering RNA(s)are 31 nucleotides in length. In some embodiments, the interferingRNA(s) are 32 nucleotides in length. In some embodiments, theinterfering RNA(s) are 33 nucleotides in length. In some embodiments,the interfering RNA(s) are 34 nucleotides in length. In someembodiments, the interfering RNA(s) are 35 nucleotides in length.

In some embodiments, the interfering RNA(s) contain a portion thatanneals to an endogenous RNA transcript containing an expanded repeatregion. The portion of each interfering RNA(s) may anneal to a segmentof the endogenous RNA transcript that does not overlap with the expandedrepeat region.

In some embodiments, the endogenous RNA transcript encodes human DMPKand contains an expanded repeat region. The expanded repeat region maycontain, for example, 50 or more CUG trinucleotide repeats, such as fromabout 50 to about 4,000 CUG trinucleotide repeats (e.g., about 50 CUGtrinucleotide repeats, about 60 CUG trinucleotide repeats, about 70trinucleotide repeats, 80 trinucleotide repeats, 90 trinucleotiderepeats, 100 trinucleotide repeats, 110 trinucleotide repeats, 120trinucleotide repeats, 130 trinucleotide repeats, 140 trinucleotiderepeats, 150 trinucleotide repeats, 160 trinucleotide repeats, 170trinucleotide repeats, 180 trinucleotide repeats, 190 trinucleotiderepeats, 200 trinucleotide repeats, 210 trinucleotide repeats, 220trinucleotide repeats, 230 trinucleotide repeats, 240 trinucleotiderepeats, 250 trinucleotide repeats, 260 trinucleotide repeats, 270trinucleotide repeats, 280 trinucleotide repeats, 290 trinucleotiderepeats, 300 trinucleotide repeats, 310 trinucleotide repeats, 320trinucleotide repeats, 330 trinucleotide repeats, 340 trinucleotiderepeats, 350 trinucleotide repeats, 360 trinucleotide repeats, 370trinucleotide repeats, 380 trinucleotide repeats, 390 trinucleotiderepeats, 400 trinucleotide repeats, 410 trinucleotide repeats, 420trinucleotide repeats, 430 trinucleotide repeats, 440 trinucleotiderepeats, 450 trinucleotide repeats, 460 trinucleotide repeats, 470trinucleotide repeats, 480 trinucleotide repeats, 490 trinucleotiderepeats, 500 trinucleotide repeats, 510 trinucleotide repeats, 520trinucleotide repeats, 530 trinucleotide repeats, 540 trinucleotiderepeats, 550 trinucleotide repeats, 560 trinucleotide repeats, 570trinucleotide repeats, 580 trinucleotide repeats, 590 trinucleotiderepeats, 600 trinucleotide repeats, 610 trinucleotide repeats, 620trinucleotide repeats, 630 trinucleotide repeats, 640 trinucleotiderepeats, 650 trinucleotide repeats, 660 trinucleotide repeats, 670trinucleotide repeats, 680 trinucleotide repeats, 690 trinucleotiderepeats, 700 trinucleotide repeats, 710 trinucleotide repeats, 720trinucleotide repeats, 730 trinucleotide repeats, 740 trinucleotiderepeats, 750 trinucleotide repeats, 760 trinucleotide repeats, 770trinucleotide repeats, 780 trinucleotide repeats, 790 trinucleotiderepeats, 800 trinucleotide repeats, 810 trinucleotide repeats, 820trinucleotide repeats, 830 trinucleotide repeats, 840 trinucleotiderepeats, 850 trinucleotide repeats, 860 trinucleotide repeats, 870trinucleotide repeats, 880 trinucleotide repeats, 890 trinucleotiderepeats, 900 trinucleotide repeats, 910 trinucleotide repeats, 920trinucleotide repeats, 930 trinucleotide repeats, 940 trinucleotiderepeats, 950 trinucleotide repeats, 960 trinucleotide repeats, 970trinucleotide repeats, 980 trinucleotide repeats, 990 trinucleotiderepeats, 1,000 trinucleotide repeats, 1,100 trinucleotide repeats, 1,200trinucleotide repeats, 1,300 trinucleotide repeats, 1,400 trinucleotiderepeats, 1,500 trinucleotide repeats, 1,600 trinucleotide repeats, 1,700trinucleotide repeats, 1,800 trinucleotide repeats, 1,900 trinucleotiderepeats, 2,000 trinucleotide repeats, 2,100 trinucleotide repeats, 2,200trinucleotide repeats, 2,300 trinucleotide repeats, 2,400 trinucleotiderepeats, 2,500 trinucleotide repeats, 2,600 trinucleotide repeats, 2,700trinucleotide repeats, 2,800 trinucleotide repeats, 2,900 trinucleotiderepeats, 3,000 trinucleotide repeats, 3,100 trinucleotide repeats, 3,200trinucleotide repeats, 3,300 trinucleotide repeats, 3,400 trinucleotiderepeats, 3,500 trinucleotide repeats, 3,600 trinucleotide repeats, 3,700trinucleotide repeats, 3,800 trinucleotide repeats, 3,900 trinucleotiderepeats, or 4,000 trinucleotide repeats, among others). In someembodiments, the endogenous RNA transcript contains a portion having atleast 85% sequence identity (e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequenceidentity) to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.In some embodiments, the endogenous RNA transcript contains a portionhaving at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to thenucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In someembodiments, the endogenous RNA transcript contains a portion having atleast 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or100% sequence identity) to the nucleic acid sequence of SEQ ID NO: 1 orSEQ ID NO: 2. The endogenous RNA transcript may contain, for example, aportion having the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.

In some embodiments, the viral vector further comprises a transgeneencoding human DMPK, such as a codon-optimized human DMPK that, upontranscription, does not anneal to the interfering RNA. For example, theDMPK transcript expressed by the transgene encoding human DMPK may beless than 85% complementary to the interfering RNA (e.g., less than 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, 5%, 4%, 3%, 2%, or 1% complementary, or less). The transgeneencoding human DMPK may be operably linked to the transgene(s) encodingthe interfering RNA, for example, such that the interfering RNA(s) andthe DMPK are expressed from the same promoter. This may be effectuated,for instance, by the placement of an internal ribosomal entry site(IRES) between the transgene(s) encoding the interfering RNA(s) and thetransgene encoding human DMPK. In some embodiments, the transgene(s)encoding the interfering RNA(s) and the transgene encoding human DMPKare each operably linked to separate promoters.

In some embodiments, the portion of each interfering RNA anneals to asegment of the endogenous RNA transcript having the nucleic acidsequence of any one of SEQ ID NOs: 3-39.

In some embodiments, the portion of each interfering RNA anneals to asegment of the endogenous RNA transcript within any one of exons 1-15 ofhuman DMPK RNA (e.g., to a segment within exon 1, exon 2, exon 3, exon4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12,exon 13, exon 14, or exon 15 of human DMPK RNA). The portion of eachinterfering RNA may have, for example, a nucleic acid sequence that isleast 85% complementary (e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% complementary) to thenucleic acid sequence of a segment within any one of exons 1-15 of humanDMPK. In some embodiments, the portion of each interfering RNA has anucleic acid sequence that is at least 90% complementary (e.g., 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment within any oneof exons 1-15 of human DMPK. For example, the portion of eachinterfering RNA may have a nucleic acid sequence that is at least 95%complementary (e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment within any oneof exons 1-15 of human DMPK. In some embodiments, the portion of eachinterfering RNA has a nucleic acid sequence that is completelycomplementary to the nucleic acid sequence of a segment within any oneof exons 1-15 of human DMPK.

In some embodiments, the portion of each interfering RNA anneals to asegment of the endogenous RNA transcript within any one of introns 1-14of human DMPK RNA (e.g., to a segment within intron 1, intron 2, intron3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron10, intron 11, intron 12, intron 13, or intron 14 of human DMPK RNA).The portion of each interfering RNA may have, for example, a nucleicacid sequence that is least 85% complementary (e.g., 85%, 85%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment within any oneof introns 1-14 human DMPK. In some embodiments, the portion of eachinterfering RNA has a nucleic acid sequence that is at least 90%complementary (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.9%, or 100% complementary) to the nucleic acid sequence of a segmentwithin any one of introns 1-14 of human DMPK. For example, the portionof each interfering RNA may have a nucleic acid sequence that is atleast 95% complementary (e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment within any oneof introns 1-14 of human DMPK. In some embodiments, the portion of eachinterfering RNA has a nucleic acid sequence that is completelycomplementary to the nucleic acid sequence of a segment within any oneof introns 1-14 of human DMPK.

In some embodiments, the portion of each interfering RNA anneals to asegment of the endogenous RNA transcript containing an exon-intronboundary within human DMPK (e.g., to a segment containing the boundarybetween exon 1 and intron 1, between intron 1 and exon 2, between exon 2and intron 2, between intron 2 and exon 3, between exon 3 and intron 3,between intron 3 and exon 4, between exon 4 and intron 4, between intron4 and exon 5, between exon 5 and intron 5, between intron 5 and exon 6,between exon 6 and intron 6, between intron 6 and exon 7, between exon 7and intron 7, between intron 7 and exon 8, between exon 8 and intron 8,between intron 8 and exon 9, between exon 9 and intron 9, between intron9 and exon 10, between exon 10 and intron 10, between intron 10 and exon11, between exon 11 and intron 11, between intron 11 and exon 12,between exon 12 and intron 12, between intron 12 and exon 13, betweenexon 13 and intron 13, between intron 13 and exon 14, between exon 14and intron 14, or between intron 14 and exon 15). The portion of eachinterfering RNA may have, for example, a nucleic acid sequence that isleast 85% complementary (e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% complementary) to thenucleic acid sequence of a segment containing an exon-intron boundarywithin human DMPK. In some embodiments, the portion of each interferingRNA has a nucleic acid sequence that is at least 90% complementary(e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment containing anexon-intron boundary within human DMPK. For example, the portion of eachinterfering RNA may have a nucleic acid sequence that is at least 95%complementary (e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment containing anexon-intron boundary within human DMPK. In some embodiments, the portionof each interfering RNA has a nucleic acid sequence that is completelycomplementary to the nucleic acid sequence of a segment containing anexon-intron boundary within human DMPK.

In some embodiments, the portion of each interfering RNA anneals to asegment of the endogenous RNA transcript within the 5′ UTR or 3′ UTR ofhuman DMPK. The portion of each interfering RNA may have, for example, anucleic acid sequence that is least 85% complementary (e.g., 85%, 85%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%,or 100% complementary) to the nucleic acid sequence of a segment withinthe 5′ UTR or 3′ UTR of human DMPK. In some embodiments, the portion ofeach interfering RNA has a nucleic acid sequence that is at least 90%complementary (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.9%, or 100% complementary) to the nucleic acid sequence of a segmentwithin the 5′ UTR or 3′ UTR of human DMPK. For example, the portion ofeach interfering RNA may have a nucleic acid sequence that is at least95% complementary (e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment within the 5′UTR or 3′ UTR of human DMPK. In some embodiments, the portion of eachinterfering RNA has a nucleic acid sequence that is completelycomplementary to the nucleic acid sequence of a segment within the 5′UTR or 3′ UTR of human DMPK.

In some embodiments, the segment within human DMPK is from about 10 toabout 80 nucleotides in length. For example, the segment may be 10nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78nucleotides, 79 nucleotides, or 80 nucleotides in length. In someembodiments, the segment within human DMPK is from about 15 to about 50nucleotides in length, such as a segment of 15 nucleotides, 16nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48nucleotides, 49 nucleotides, or 50 nucleotides in length. In someembodiments, the segment within human DMPK is from about 17 to about 23nucleotides in length, such as 17 nucleotides, 18 nucleotides, 19nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23nucleotides in length. In some embodiments, the segment is 18nucleotides in length. In some embodiments, the segment is 19nucleotides in length. In some embodiments, the segment is 20nucleotides in length. In some embodiments, the segment is 21nucleotides in length.

In some embodiments, the interfering RNA anneals to the endogenous RNAtranscript encoding human DMPK with from one to eight nucleotidemismatches (e.g., with one nucleotide mismatch, two nucleotidemismatches, three nucleotide mismatches, four nucleotide mismatches,five nucleotide mismatches, six nucleotide mismatches, seven nucleotidemismatches, or eight nucleotide mismatches). In some embodiments, theinterfering RNA anneals to the endogenous RNA transcript encoding humanDMPK with from one to five nucleotide mismatches, such as with onenucleotide mismatch, two nucleotide mismatches, three nucleotidemismatches, four nucleotide mismatches, or five nucleotide mismatches.In some embodiments, the interfering RNA anneals to the endogenous RNAtranscript encoding human DMPK with from one to three nucleotidemismatches, such as with one nucleotide mismatch, two nucleotidemismatches, or three nucleotide mismatches. In some embodiments, theinterfering RNA anneals to the endogenous RNA transcript encoding humanDMPK with no more than two nucleotide mismatches. For example,interfering RNA may anneal to the endogenous RNA transcript encodinghuman DMKPK with no nucleotide mismatches, one nucleotide mismatch, ortwo nucleotide mismatches.

In some embodiments, the interfering RNA contains a portion having atleast 85% sequence identity (e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequenceidentity) to the nucleic acid sequence of any one of SEQ ID NOs: 3-161.The interfering RNA may contain, for example, a portion having at least90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.9%, or 100% sequence identity) to the nucleic acid sequenceof ay one of SEQ ID NOs: 3-161. In some embodiments, the interfering RNAcontains a portion having at least 95% sequence identity (e.g., 95%,96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to the nucleicacid sequence of ay one of SEQ ID NOs: 3-161. In some embodiments, theinterfering RNA contains a portion having the nucleic acid sequence ofany one of SEQ ID NOs: 3-161. In some embodiments, the interfering RNAis a miRNA having a combination of passenger and guide strands shown inTable 5, herein.

In some embodiments, the endogenous RNA transcript contains humanchromosome 9 open reading frame 72 (C9ORF72) and an expanded repeatregion. The expanded repeat region may contain, for example, greaterthan 25 GGGGCC (SEQ ID NO: 162) hexanucleotide repeats, such as fromabout 700 to about 1,600 GGGGCC (SEQ ID NO: 162) hexanucleotide repeats,For example, the expanded repeat region may contain 700 hexanucleotiderepeats, 710 hexanucleotide repeats, 720 hexanucleotide repeats, 730hexanucleotide repeats, 740 hexanucleotide repeats, 750 hexanucleotiderepeats, 760 hexanucleotide repeats, 770 hexanucleotide repeats, 780hexanucleotide repeats, 790 hexanucleotide repeats, 800 hexanucleotiderepeats, 810 hexanucleotide repeats, 820 hexanucleotide repeats, 830hexanucleotide repeats, 840 hexanucleotide repeats, 850 hexanucleotiderepeats, 860 hexanucleotide repeats, 870 hexanucleotide repeats, 880hexanucleotide repeats, 890 hexanucleotide repeats, 900 hexanucleotiderepeats, 910 hexanucleotide repeats, 920 hexanucleotide repeats, 930hexanucleotide repeats, 940 hexanucleotide repeats, 950 hexanucleotiderepeats, 960 hexanucleotide repeats, 970 hexanucleotide repeats, 980hexanucleotide repeats, 990 hexanucleotide repeats, or 1,000hexanucleotide repeats, among others. The expanded repeat region maycontain greater than 30 hexanucleotide repeats, such as 30hexanucleotide repeats, 40 hexanucleotide repeats, 50 hexanucleotiderepeats, 60 CUG hexanucleotide repeats, 70 hexanucleotide repeats, 80hexanucleotide repeats, 90 hexanucleotide repeats, 100 hexanucleotiderepeats, 110 hexanucleotide repeats, 120 hexanucleotide repeats, 130hexanucleotide repeats, 140 hexanucleotide repeats, 150 hexanucleotiderepeats, 160 hexanucleotide repeats, 170 hexanucleotide repeats, 180hexanucleotide repeats, 190 hexanucleotide repeats, 200 hexanucleotiderepeats, 210 hexanucleotide repeats, 220 hexanucleotide repeats, 230hexanucleotide repeats, 240 hexanucleotide repeats, 250 hexanucleotiderepeats, 260 hexanucleotide repeats, 270 hexanucleotide repeats, 280hexanucleotide repeats, 290 hexanucleotide repeats, 300 hexanucleotiderepeats, 310 hexanucleotide repeats, 320 hexanucleotide repeats, 330hexanucleotide repeats, 340 hexanucleotide repeats, 350 hexanucleotiderepeats, 360 hexanucleotide repeats, 370 hexanucleotide repeats, 380hexanucleotide repeats, 390 hexanucleotide repeats, 400 hexanucleotiderepeats, 410 hexanucleotide repeats, 420 hexanucleotide repeats, 430hexanucleotide repeats, 440 hexanucleotide repeats, 450 hexanucleotiderepeats, 460 hexanucleotide repeats, 470 hexanucleotide repeats, 480hexanucleotide repeats, 490 hexanucleotide repeats, 500 hexanucleotiderepeats, 510 hexanucleotide repeats, 520 hexanucleotide repeats, 530hexanucleotide repeats, 540 hexanucleotide repeats, 550 hexanucleotiderepeats, 560 hexanucleotide repeats, 570 hexanucleotide repeats, 580hexanucleotide repeats, 590 hexanucleotide repeats, 600 hexanucleotiderepeats, 610 hexanucleotide repeats, 620 hexanucleotide repeats, 630hexanucleotide repeats, 640 hexanucleotide repeats, 650 hexanucleotiderepeats, 660 hexanucleotide repeats, 670 hexanucleotide repeats, 680hexanucleotide repeats, 690 hexanucleotide repeats, 700 hexanucleotiderepeats, 710 hexanucleotide repeats, 720 hexanucleotide repeats, 730hexanucleotide repeats, 740 hexanucleotide repeats, 750 hexanucleotiderepeats, 760 hexanucleotide repeats, 770 hexanucleotide repeats, 780hexanucleotide repeats, 790 hexanucleotide repeats, 800 hexanucleotiderepeats, 810 hexanucleotide repeats, 820 hexanucleotide repeats, 830hexanucleotide repeats, 840 hexanucleotide repeats, 850 hexanucleotiderepeats, 860 hexanucleotide repeats, 870 hexanucleotide repeats, 880hexanucleotide repeats, 890 hexanucleotide repeats, 900 hexanucleotiderepeats, 910 hexanucleotide repeats, 920 hexanucleotide repeats, 930hexanucleotide repeats, 940 hexanucleotide repeats, 950 hexanucleotiderepeats, 960 hexanucleotide repeats, 970 hexanucleotide repeats, 980hexanucleotide repeats, 990 hexanucleotide repeats, 1,000 hexanucleotiderepeats, 1,100 hexanucleotide repeats, 1,200 hexanucleotide repeats,1,300 hexanucleotide repeats, 1,400 hexanucleotide repeats, 1,500hexanucleotide repeats, 1,600 hexanucleotide repeats, or more.

In some embodiments, the endogenous RNA transcript contains a portionhaving at least 85% sequence identity (e.g., 85%, 85%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%sequence identity) to the nucleic acid sequence of SEQ ID NO: 163. Theendogenous RNA transcript may contain, for example, a portion having atleast 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.9%, or 100% sequence identity) to the nucleic acidsequence of SEQ ID NO: 163. In some embodiments, the endogenous RNAtranscript contains a portion having at least 95% sequence identity(e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to thenucleic acid sequence of SEQ ID NO: 163. In some embodiments, the RNAtranscript contains a portion having the nucleic acid sequence of SEQ IDNO: 163.

In some embodiments, the endogenous RNA transcript contains a portionhaving at least 85% sequence identity (e.g., 85%, 85%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%sequence identity) to the nucleic acid sequence of SEQ ID NO: 165. Theendogenous RNA transcript may contain, for example, a portion having atleast 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.9%, or 100% sequence identity) to the nucleic acidsequence of SEQ ID NO: 165. In some embodiments, the endogenous RNAtranscript contains a portion having at least 95% sequence identity(e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to thenucleic acid sequence of SEQ ID NO: 165. In some embodiments, the RNAtranscript contains a portion having the nucleic acid sequence of SEQ IDNO: 165.

In some embodiments, the endogenous RNA transcript contains a portionhaving at least 85% sequence identity (e.g., 85%, 85%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%sequence identity) to the nucleic acid sequence of SEQ ID NO: 166. Theendogenous RNA transcript may contain, for example, a portion having atleast 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.9%, or 100% sequence identity) to the nucleic acidsequence of SEQ ID NO: 166. In some embodiments, the endogenous RNAtranscript contains a portion having at least 95% sequence identity(e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to thenucleic acid sequence of SEQ ID NO: 166. In some embodiments, the RNAtranscript contains a portion having the nucleic acid sequence of SEQ IDNO: 166.

In some embodiments, the portion of each interfering RNA has a nucleicacid sequence that is least 85% complementary (e.g., 85%, 85%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment within humanC9ORF72, such as to the nucleic acid sequence of a segment within SEQ IDNO: 163, 165, or 166. In some embodiments, the portion of eachinterfering RNA has a nucleic acid sequence that is at least 90%complementary (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.9%, or 100% complementary) to the nucleic acid sequence of a segmentwithin human C9ORF72, such as to the nucleic acid sequence of a segmentwithin SEQ ID NO: 163, 165, or 166. For example, the portion of eachinterfering RNA may have a nucleic acid sequence that is at least 95%complementary (e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment within humanC9ORF72, such as to the nucleic acid sequence of a segment within SEQ IDNO: 163, 165, or 166. In some embodiments, the portion of eachinterfering RNA has a nucleic acid sequence that is completelycomplementary to the nucleic acid sequence of a segment within humanC9ORF72, such as to the nucleic acid sequence of a segment within SEQ IDNO: 163, 165, or 166.

In some embodiments, the segment within human C9ORF72 is from about 10to about 80 nucleotides in length. For example, the segment may be 10nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78nucleotides, 79 nucleotides, or 80 nucleotides in length. In someembodiments, the segment within human C9ORF72 is from about 15 to about50 nucleotides in length, such as a segment of 15 nucleotides, 16nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48nucleotides, 49 nucleotides, or 50 nucleotides in length. In someembodiments, the segment within human C9ORF72 is from about 17 to about23 nucleotides in length, such as 17 nucleotides, 18 nucleotides, 19nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23nucleotides in length. In some embodiments, the segment is 18nucleotides in length. In some embodiments, the segment is 19nucleotides in length. In some embodiments, the segment is 20nucleotides in length. In some embodiments, the segment is 21nucleotides in length.

In some embodiments, the interfering RNA anneals to the endogenous RNAtranscript encoding human C9ORF72 with from one to eight nucleotidemismatches (e.g., with one nucleotide mismatch, two nucleotidemismatches, three nucleotide mismatches, four nucleotide mismatches,five nucleotide mismatches, six nucleotide mismatches, seven nucleotidemismatches, or eight nucleotide mismatches). In some embodiments, theinterfering RNA anneals to the endogenous RNA transcript encoding humanC9ORF72 with from one to five nucleotide mismatches, such as with onenucleotide mismatch, two nucleotide mismatches, three nucleotidemismatches, four nucleotide mismatches, or five nucleotide mismatches.In some embodiments, the interfering RNA anneals to the endogenous RNAtranscript encoding human C9ORF72 with from one to three nucleotidemismatches, such as with one nucleotide mismatch, two nucleotidemismatches, or three nucleotide mismatches. In some embodiments, theinterfering RNA anneals to the endogenous RNA transcript encoding humanC9ORF72 with no more than two nucleotide mismatches. For example,interfering RNA may anneal to the endogenous RNA transcript encodinghuman C9ORF72 with no nucleotide mismatches, one nucleotide mismatch, ortwo nucleotide mismatches.

In some embodiments, the interfering RNA is a short interfering RNA(siRNA), a short hairpin RNA (shRNA), or a micro RNA (miRNA), such as aU6 miRNA. The miRNA may be based, for example, on the endogenous humanmiR30a nucleic acid sequence, having one or more nucleic acidsubstitutions as needed for complementarity to a target mRNA (e.g., atarget mRNA described herein). In the case of a miRNA, the viral vectormay contain, for example, a primary miRNA (pri-miRNA) transcriptencoding a mature miRNA. In some embodiments, the viral vector containsa pre-miRNA transcript encoding a mature miRNA.

In some embodiments, the interfering RNA is operably linked to apromoter that induces expression of the interfering RNA in a muscle cellor neuron. The promoter may be, for example, a desmin promoter, aphosphoglycerate kinase (PGK) promoter, a muscle creatine kinasepromoter, a myosin light chain promoter, a myosin heavy chain promoter,a cardiac troponin C promoter, a troponin I promoter, a myoD gene familypromoter, an actin alpha promoter, an actin beta promoter, an actingamma promoter, or a promoter within intron 1 of ocular paired likehomeodomain 3 (PITX3).

In some embodiments, the viral vector is an AAV, adenovirus, lentivirus,retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus,or a synthetic virus. The viral vector may be, for example, an AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74serotype. In some embodiments, the viral vector is a pseudotyped AAV,such as an AAV2/8 or AAV/29 vector. The viral vector may contain arecombinant capsid protein. In some embodiments, the viral vector is asynthetic virus, such as a chimeric virus, mosaic virus, or pseudotypedvirus, and/or a synthetic virus that contains a foreign protein,synthetic polymer, nanoparticle, or small molecule.

In another aspect, the invention features a nucleic acid encoding orcontaining an interfering RNA that contains a portion having at least85% sequence identity (e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) tothe nucleic acid sequence of any one of SEQ ID NOs: 3-161. In someembodiments, the interfering RNA contains a portion having at least 90%sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% sequence identity) to the nucleic acid sequence ofany one of SEQ ID NOs: 3-161. The interfering RNA may contain, forexample, a portion having at least 95% sequence identity (e.g., 95%,96%, 97%, 98%, 99%, 99.9%, or 100% sequence identity) to the nucleicacid sequence of any one of SEQ ID NOs: 3-161. In some embodiments, theinterfering RNA contains a portion having the nucleic acid sequence ofany one of SEQ ID NOs: 3-161. In some embodiments, the interfering RNAis a miRNA having a combination of passenger and guide strands shown inTable 5, herein.

In some embodiments, the portion of each interfering RNA anneals to asegment of an endogenous RNA transcript encoding human DMPK within anyone of exons 1-15 of human DMPK RNA (e.g., to a segment within exon 1,exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10,exon 11, exon 12, exon 13, exon 14, or exon 15 of human DMPK RNA). Theportion of each interfering RNA may have, for example, a nucleic acidsequence that is least 85% complementary (e.g., 85%, 85%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment within any oneof exons 1-15 of human DMPK. In some embodiments, the portion of eachinterfering RNA has a nucleic acid sequence that is at least 90%complementary (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.9%, or 100% complementary) to the nucleic acid sequence of a segmentwithin any one of exons 1-15 of human DMPK. For example, the portion ofeach interfering RNA may have a nucleic acid sequence that is at least95% complementary (e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment within any oneof exons 1-15 of human DMPK. In some embodiments, the portion of eachinterfering RNA has a nucleic acid sequence that is completelycomplementary to the nucleic acid sequence of a segment within any oneof exons 1-15 of human DMPK.

In some embodiments, the portion of each interfering RNA anneals to asegment of an endogenous RNA transcript encoding human DMPK within anyone of introns 1-14 of human DMPK RNA (e.g., to a segment within intron1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8,intron 9, intron 10, intron 11, intron 12, intron 13, or intron 14 ofhuman DMPK RNA). The portion of each interfering RNA may have, forexample, a nucleic acid sequence that is least 85% complementary (e.g.,85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.9%, or 100% complementary) to the nucleic acid sequence of asegment within any one of introns 1-14 human DMPK. In some embodiments,the portion of each interfering RNA has a nucleic acid sequence that isat least 90% complementary (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.9%, or 100% complementary) to the nucleic acidsequence of a segment within any one of introns 1-14 of human DMPK. Forexample, the portion of each interfering RNA may have a nucleic acidsequence that is at least 95% complementary (e.g., 95%, 96%, 97%, 98%,99%, 99.9%, or 100% complementary) to the nucleic acid sequence of asegment within any one of introns 1-14 of human DMPK. In someembodiments, the portion of each interfering RNA has a nucleic acidsequence that is completely complementary to the nucleic acid sequenceof a segment within any one of introns 1-14 of human DMPK.

In some embodiments, the portion of each interfering RNA anneals to asegment of an endogenous RNA transcript encoding human DMPK containingan exon-intron boundary within human DMPK (e.g., to a segment containingthe boundary between exon 1 and intron 1, between intron 1 and exon 2,between exon 2 and intron 2, between intron 2 and exon 3, between exon 3and intron 3, between intron 3 and exon 4, between exon 4 and intron 4,between intron 4 and exon 5, between exon 5 and intron 5, between intron5 and exon 6, between exon 6 and intron 6, between intron 6 and exon 7,between exon 7 and intron 7, between intron 7 and exon 8, between exon 8and intron 8, between intron 8 and exon 9, between exon 9 and intron 9,between intron 9 and exon 10, between exon 10 and intron 10, betweenintron 10 and exon 11, between exon 11 and intron 11, between intron 11and exon 12, between exon 12 and intron 12, between intron 12 and exon13, between exon 13 and intron 13, between intron 13 and exon 14,between exon 14 and intron 14, or between intron 14 and exon 15). Theportion of each interfering RNA may have, for example, a nucleic acidsequence that is least 85% complementary (e.g., 85%, 85%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%complementary) to the nucleic acid sequence of a segment containing anexon-intron boundary within human DMPK. In some embodiments, the portionof each interfering RNA has a nucleic acid sequence that is at least 90%complementary (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.9%, or 100% complementary) to the nucleic acid sequence of a segmentcontaining an exon-intron boundary within human DMPK. For example, theportion of each interfering RNA may have a nucleic acid sequence that isat least 95% complementary (e.g., 95%, 96%, 97%, 98%, 99%, 99.9%, or100% complementary) to the nucleic acid sequence of a segment containingan exon-intron boundary within human DMPK. In some embodiments, theportion of each interfering RNA has a nucleic acid sequence that iscompletely complementary to the nucleic acid sequence of a segmentcontaining an exon-intron boundary within human DMPK.

In some embodiments, the portion of each interfering RNA anneals to asegment of an endogenous RNA transcript encoding human DMPK within the5′ UTR or 3′ UTR of human DMPK. The portion of each interfering RNA mayhave, for example, a nucleic acid sequence that is least 85%complementary (e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.9%, or 100% complementary) to the nucleicacid sequence of a segment within the 5′ UTR or 3′ UTR of human DMPK. Insome embodiments, the portion of each interfering RNA has a nucleic acidsequence that is at least 90% complementary (e.g., 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% complementary) to thenucleic acid sequence of a segment within the 5′ UTR or 3′ UTR of humanDMPK. For example, the portion of each interfering RNA may have anucleic acid sequence that is at least 95% complementary (e.g., 95%,96%, 97%, 98%, 99%, 99.9%, or 100% complementary) to the nucleic acidsequence of a segment within the 5′ UTR or 3′ UTR of human DMPK. In someembodiments, the portion of each interfering RNA has a nucleic acidsequence that is completely complementary to the nucleic acid sequenceof a segment within the 5′ UTR or 3′ UTR of human DMPK.

In some embodiments, the segment within human DMPK is from about 10 toabout 80 nucleotides in length. For example, the segment may be 10nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78nucleotides, 79 nucleotides, or 80 nucleotides in length. In someembodiments, the segment within human DMPK is from about 15 to about 50nucleotides in length, such as a segment of 15 nucleotides, 16nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48nucleotides, 49 nucleotides, or 50 nucleotides in length. In someembodiments, the segment within human DMPK is from about 17 to about 23nucleotides in length, such as 17 nucleotides, 18 nucleotides, 19nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23nucleotides in length. In some embodiments, the segment is 18nucleotides in length. In some embodiments, the segment is 19nucleotides in length. In some embodiments, the segment is 20nucleotides in length. In some embodiments, the segment is 21nucleotides in length.

In some embodiments, the interfering RNA anneals to an endogenous RNAtranscript encoding human DMPK with from one to eight nucleotidemismatches (e.g., with one nucleotide mismatch, two nucleotidemismatches, three nucleotide mismatches, four nucleotide mismatches,five nucleotide mismatches, six nucleotide mismatches, seven nucleotidemismatches, or eight nucleotide mismatches). In some embodiments, theinterfering RNA anneals to an endogenous RNA transcript encoding humanDMPK with from one to five nucleotide mismatches, such as with onenucleotide mismatch, two nucleotide mismatches, three nucleotidemismatches, four nucleotide mismatches, or five nucleotide mismatches.In some embodiments, the interfering RNA anneals to an endogenous RNAtranscript encoding human DMPK with from one to three nucleotidemismatches, such as with one nucleotide mismatch, two nucleotidemismatches, or three nucleotide mismatches. In some embodiments, theinterfering RNA anneals to an endogenous RNA transcript encoding humanDMPK with no more than two nucleotide mismatches. For example,interfering RNA may anneal to the endogenous RNA transcript encodinghuman DMKPK with no nucleotide mismatches, one nucleotide mismatch, ortwo nucleotide mismatches.

In some embodiments, the interfering RNA is a siRNA, a shRNA, or amiRNA, such as a U6 miRNA. The miRNA may be based, for example, on theendogenous human miR30a nucleic acid sequence, having one or morenucleic acid substitutions as needed for complementarity to a targetmRNA (e.g., a target mRNA described herein). In the case of a miRNA, thenucleic acid may contain, for example, a pri-miRNA transcript encoding amature miRNA. In some embodiments, the viral vector contains a pre-miRNAtranscript encoding a mature miRNA.

In some embodiments, the interfering RNA is operably linked to apromoter that induces expression of the interfering RNA in a muscle cellor neuron. The promoter may be, for example, a desmin promoter, a PGKpromoter, a muscle creatine kinase promoter, a myosin light chainpromoter, a myosin heavy chain promoter, a cardiac troponin C promoter,a troponin I promoter, a myoD gene family promoter, an actin alphapromoter, an actin beta promoter, an actin gamma promoter, or a promoterwithin intron 1 of ocular PITX3.

In an additional aspect, the invention features a vector containing thenucleic acid of any of the above aspects or embodiments. The vector maybe, for example, an AAV (e.g., an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAVrh10, or AAVrh74 serotype, or a pseudotyped AAV,such as an AAV2/8 or AAV/29 vector), adenovirus, lentivirus, retrovirus,poxvirus, baculovirus, herpes simplex virus, vaccinia virus, or asynthetic virus (e.g., a chimeric virus, mosaic virus, or pseudotypedvirus, and/or a synthetic virus that contains a foreign protein,synthetic polymer, nanoparticle, or small molecule), and may contain oneor more recombinant capsid proteins.

In yet another aspect, the invention features a composition containingthe nucleic acid of any of the above aspects or embodiments. Thecomposition may be, for example, a liposome, vesicle, synthetic vesicle,exosome, synthetic exosome, dendrimer, or nanoparticle.

In another aspect, the invention features a pharmaceutical compositioncontaining the nucleic acid of any of the above aspects or embodiments.The pharmaceutical composition may further contain a pharmaceuticallyacceptable carrier, diluent, or excipient.

In an additional aspect, the invention features a method of reducing theoccurrence of spliceopathy (e.g., of an mRNA transcript for whichsplicing is regulated, in part, by the activity of muscleblind-likeprotein) in a patient, such as a human patient, in need thereof. Themethod may include administering to the patient a therapeuticallyeffective amount of the vector or composition of any of the aboveaspects or embodiments. In some embodiments, the patient has myotonicdystrophy. Upon administration of the vector or composition to thepatient, the patient may exhibit an increase in corrective splicing ofone or more RNA transcript substrates of muscleblind-like protein.

In another aspect, the invention features a method of treating adisorder characterized by nuclear retention of RNA containing anexpanded repeat region in a patient, such as a human patient, in needthereof by administering to the patient a therapeutically effectiveamount of the vector or composition of any of the above aspects orembodiments. The disorder may be, for example, myotonic dystrophy, andthe nuclear-retained RNA may be DMPK RNA. In some embodiments, thedisorder is amyotrophic lateral sclerosis and the nuclear-retrained RNAis C9ORF72 RNA.

Upon administration of the vector or composition to the patient, thepatient may exhibit an increase in corrective splicing of one or moreRNA transcript substrates of muscleblind-like protein. For example, uponadministration of the vector or composition to the patient, the patientmay exhibit an increase in expression of sarcoplasmic/endoplasmicreticulum calcium ATPase 1 (SERCA1) mRNA containing exon 22, such as anincrease of about 1.1-fold to about 10-fold, or more (e.g., an increasein expression of SERCA1 mRNA containing exon 22 by about 1.1-fold,1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold,2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold,3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold,4-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold,4.7-fold, 4.8-fold, 4.9-fold, 5-fold, 5.1-fold, 5.2-fold, 5.3-fold,5.4-fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6-fold,6.1-fold, 6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold, 6.6-fold, 6.7-fold,6.8-fold, 6.9-fold, 7-fold, 7.1-fold, 7.2-fold, 7.3-fold, 7.4-fold,7.5-fold, 7.6-fold, 7.7-fold, 7.8-fold, 7.9-fold, 8-fold, 8.1-fold,8.2-fold, 8.3-fold, 8.4-fold, 8.5-fold, 8.6-fold, 8.7-fold, 8.8-fold,8.9-fold, 9-fold, 9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold,9.6-fold, 9.7-fold, 9.8-fold, 9.9-fold, 10-fold, or more), as assessed,for example, using an RNA or protein detection assay described herein.

In some embodiments, upon administration of the vector or composition tothe patient, the patient may exhibit a decrease in expression ofchloride voltage-gated channel 1 (CLCN1) mRNA containing exon 7a, suchas a decrease of about 1% to about 100% (e.g., a decrease in expressionof CLCN1 mRNA containing exon 7a by about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), as assessed, for example,using an RNA or protein detection assay described herein.

For example, upon administration of the vector or composition to thepatient, the patient may exhibit a decrease in expression of ZO-2associated speckle protein (ZASP) containing exon 11, such as a decreaseof about 1% to about 100% (e.g., a decrease in expression of ZASP mRNAcontaining exon 11 by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%), as assessed, for example, using anRNA or protein detection assay described herein.

In some embodiments, upon administration of the vector or composition tothe patient, the patient exhibits an increase in corrective splicing ofRNA transcripts encoding insulin receptor, ryanodine receptor 1 (RYR1),cardiac muscle troponin, and/or skeletal muscle troponin, such as anincrease of about 1.1-fold to about 10-fold, or more (e.g., an increasein expression of correctly spliced RNA transcripts encoding insulinreceptor, RYR1, cardiac muscle troponin, and/or skeletal muscle troponinby about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold,2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold,3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold,3.8-fold, 3.9-fold, 4-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold,4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5-fold, 5.1-fold,5.2-fold, 5.3-fold, 5.4-fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold,5.9-fold, 6-fold, 6.1-fold, 6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold,6.6-fold, 6.7-fold, 6.8-fold, 6.9-fold, 7-fold, 7.1-fold, 7.2-fold,7.3-fold, 7.4-fold, 7.5-fold, 7.6-fold, 7.7-fold, 7.8-fold, 7.9-fold,8-fold, 8.1-fold, 8.2-fold, 8.3-fold, 8.4-fold, 8.5-fold, 8.6-fold,8.7-fold, 8.8-fold, 8.9-fold, 9-fold, 9.1-fold, 9.2-fold, 9.3-fold,9.4-fold, 9.5-fold, 9.6-fold, 9.7-fold, 9.8-fold, 9.9-fold, 10-fold, ormore), as assessed, for example, using an RNA or protein detection assaydescribed herein.

In some embodiments of either of the preceding two preceding aspects,the vector or composition is administered to the patient by way ofintravenous, intrathecal, intracerebroventricular, intraparenchymal,intracisternal, intradermal, transdermal, parenteral, intramuscular,intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal,intraarterial, intravascular, inhalation, perfusion, lavage, or oraladministration.

In another aspect, the invention features a kit containing the vector orcomposition of any of the above aspects or embodiments. The kit mayfurther contain a package insert instructing a user of the kit toadminister the vector or composition to the patient to reduce theoccurrence of a spliceopathy in the patient, such as spliceopathy of anmRNA transcript for which splicing is regulated, in part, by theactivity of muscleblind-like protein.

In an additional aspect, the invention features a kit containing thevector or composition of any of the above aspects or embodiments and apackage insert that instructs a user of the kit to administer the vectoror composition to the patient to reduce the occurrence of a spliceopathyin the patient to treat a disorder characterized by nuclear retention ofRNA containing an expanded repeat region. The disorder may be, forexample, myotonic dystrophy, and the nuclear-retained RNA may be DMPKRNA. In some embodiments, the disorder is amyotrophic lateral sclerosisand the nuclear-retrained RNA is C9ORF72 RNA.

Definitions

As used herein, the term “about” refers to a value that is within 10%above or below the value being described. For example, the phrase “about100 nucleic acid residues” refers to a value of from 90 to 110 nucleicacid residues.

As used herein, the term “anneal” refers to the formation of a stableduplex of nucleic acids by way of hybridization mediated by inter-strandhydrogen bonding, for example, according to Watson-Crick base pairing.The nucleic acids of the duplex may be, for example, at least 50%complementary to one another (e.g., about 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or100% complementary to one another. The “stable duplex” formed upon theannealing of one nucleic acid to another is a duplex structure that isnot denatured by a stringent wash. Exemplary stringent wash conditionsare known in the art and include temperatures of about 5° C. less thanthe melting temperature of an individual strand of the duplex and lowconcentrations of monovalent salts, such as monovalent saltconcentrations (e.g., NaCl concentrations) of less than 0.2 M (e.g., 0.2M, 0.19 M, 0.18 M, 0.17 M, 0.16 M, 0.15 M, 0.14 M, 0.13 M, 0.12 M, 0.11M, 0.1 M, 0.09 M, 0.08 M, 0.07 M, 0.06 M, 0.05 M, 0.04 M, 0.03 M, 0.02M, 0.01 M, or less).

As used herein, the terms “conservative mutation,” “conservativesubstitution,” or “conservative amino acid substitution” refer to asubstitution of one or more amino acids for one or more different aminoacids that exhibit similar physicochemical properties, such as polarity,electrostatic charge, and steric volume. These properties are summarizedfor each of the twenty naturally-occurring amino acids in Table 1 below.

TABLE 1 Representative physicochemical properties of naturally-occurringamino acids Electrostatic 3 1 Side- character at Letter Letter chainphysiological Steric Amino Acid Code Code Polarity pH (7.4) Volume^(†)Alanine Ala A nonpolar neutral small Arginine Arg R polar cationic largeAsparagine Asn N polar neutral intermediate Aspartic acid Asp D polaranionic intermediate Cysteine Cys C nonpolar neutral intermediateGlutamic acid Glu E polar anionic intermediate Glutamine Gln Q polarneutral intermediate Glycine Gly G nonpolar neutral small Histidine HisH polar Both neutral large and cationic forms in equilibrium at pH 7.4Isoleucine Ile I nonpolar neutral large Leucine Leu L nonpolar neutrallarge Lysine Lys K polar cationic large Methionine Met M nonpolarneutral large Phenylalanine Phe F nonpolar neutral large Proline Pro Pnon- neutral intermediate polar Serine Ser S polar neutral smallThreonine Thr T polar neutral intermediate Tryptophan Trp W nonpolarneutral bulky Tyrosine Tyr Y polar neutral large Valine Val V nonpolarneutral intermediate ^(†)based on volume in A³: 50-100 is small, 100-150is intermediate, 150-200 is large, and >200 is bulky

From this table it is appreciated that the conservative amino acidfamilies include, e.g., (i) G, A, V, L, I, P, and M; (ii) D and E; (iii)C, S and T; (iv) H, K and R; (v) N and Q; and (vi) F, Y and W. Aconservative mutation or substitution is therefore one that substitutesone amino acid for a member of the same amino acid family (e.g., asubstitution of Ser for Thr or Lys for Arg).

As used herein, the terms “dystrophia myotonica protein kinase” and itsabbreviation, “DMPK,” refer to the serine/threonine kinase proteininvolved in the regulation of skeletal muscle structure and function,for example, in human subjects. The terms “dystrophia myotonica proteinkinase” and “DMPK” are used interchangeably herein and refer not only towild-type forms of the DMPK gene, but also to variants of wild-type DMPKproteins and nucleic acids encoding the same. The nucleic acid sequencesof two isoforms of human DMPK mRNA are provided herein as SEQ ID NOs: 1and 2, which correspond to GenBank Accession Nos. BC026328.1 andBC062553.1, respectively (3′ UTRs not included). These nucleic acidsequences are provided in Table 2, below.

TABLE 2 Nucleic acid sequences of exemplary human DMPK isoformsSEQ ID NO. Nucleic acid sequence 1GGCUGGACCAAGGGGUGGGGAGAAGGGGAGGAGGCCUCGGCCGGCCGCAGAGAGAAGUGGCCAGAGAGGCCCAGGGGACAGCCAGGGACAGGCAGACAUGCAGCCAGGGCUCCAGGGCCUGGACAGGGGCUGCCAGGCCCUGUGACAGGAGGACCCCGAGCCCCCGGCCCGGGGAGGGGCCAUGGUGCUGCCUGUCCAACAUGUCAGCCGAGGUGCGGCUGAGGCGGCUCCAGCAGCUGGUGUUGGACCCGGGCUUCCUGGGGCUGGAGCCCCUGCUCGACCUUCUCCUGGGCGUCCACCAGGAGCUGGGCGCCUCCGAACUGGCCCAGGACAAGUACGUGGCCGACUUCUUGCAGUGGGCCCCAAAUCCAGGGUUUUCCAAAGUGUGGUUCAAGAACCACCUGCAUCUGAAUCUAGAGCGGAGCCCAUCGUGGUGAGGCUUAAGGAGGUCCGACUGCAGAGGGACGACUUCGAGAUUCUGAAGGUGAUCGGACGCGGGGCGUUCAGCGAGGUAGCGGUAGUGAAGAUGAAGCAGACGGGCCAGGUGUAUGCCAUGAAGAUCAUGAACAAGUGGGACAUGCUGAAGAGGGGCGAGGUGUCGUGCUUCCGUGAGGAGAGGGACGUGUUGGUGAAUGGGGACCAGCGGUGGAUCACGCAGCUGCACUUCGCCUUCCAGGAUGAGAACUACCUGUACCUGGUCAUGGAGUAUUACGUGGGCGGGGACCUGCUGACACUGCUGAGCAAGUUUGGGGAGCGGAUUCCGGCCGAGAUGGCGCGCUUCUACCUGGCGGAGAUUGUCAUGGCCAUAGACUCGGUGCACCGGCUUGGCUACGUGCACAGGGACAUCAAACCCGACAACAUCCUGCUGGACCGCUGUGGCCACAUCCGCCUGGCCGACUUCGGCUCUUGCCUCAAGCUGCGGGCAGAUGGAACGGUGCGGUCGCUGGUGGCUGUGGGCACCCCAGACUACCUGUCCCCCGAGAUCCUGCAGGCUGUGGGCGGUGGGCCUGGGACAGGCAGCUACGGGCCCGAGUGUGACUGGUGGGCGCUGGGUGUAUUCGCCUAUGAAAUGUUCUAUGGGCAGACGCCCUUCUACGCGGAUUCCACGGCGGAGACCUAUGGCAAGAUCGUCCACUACAAGGAGCACCUCUCUCUGCCGCUGGUGGACGAAGGGGUCCCUGAGGAGGCUCGAGACUUCAUUCAGCGGUUGCUGUGUCCCCCGGAGACACGGCUGGGCCGGGGUGGAGCAGGCGACUUCCGGACACAUCCCUUCUUCUUUGGCCUCGACUGGGAUGGUCUCCGGGACAGCGUGCCCCCCUUUACACCGGAUUUCGAAGGUGCCACCGACACAUGCAACUUCGACUUGGUGGAGGACGGGCUCACUGCCAUGGUGAGCGGGGGCGGGGAGACACUGUCGGACAUUCGGGAAGGUGCGCCGCUAGGGGUCCACCUGCCUUUUGUGGGCUACUCCUACUCCUGCAUGGCCCUCAGGGACAGUGAGGUCCCAGGCCCCACACCCAUGGAACUGGAGGCCGAGCAGCUGCUUGAGCCACACGUGCAAGCGCCCAGCCUGGAGCCCUCGGUGUCCCCACAGGAUGAAACAGCUGAAGUGGCAGUUCCAGCGGCUGUCCCUGCGGCAGAGGCUGAGGCCGAGGUGACGCUGCGGGAGCUCCAGGAAGCCCUGGAGGAGGAGGUGCUCACCCGGCAGAGCCUGAGCCGGGAGAUGGAGGCCAUCCGCACGGACAACCAGAACUUCGCCAGUCAACUACGCGAGGCAGAGGCUCGGAACCGGGACCUAGAGGCACACGUCCGGCAGUUGCAGGAGCGGAUGGAGUUGCUGCAGGCAGAGGGAGCCACAGCUGUCACGGGGGUCCCCAGUCCCCGGGCCACGGAUCCACCUUCCCAUCUAGAUGGCCCCACGGCCGUGGCUGUGGGCCAGUGCCCGCUGGUGGGGCCAGGCCCCAUGCACCGCCGCCACCUGCUGCUCCCUGCCAGGGUCCCUAGGCCUGGCCUAUCGGAGGCGCUUUCCCUGCUCCUGUUCGCCGUUGUUCUGUCUCGUGCCGCCGCCCUGGGCUGCAUUGGGUUGGUGGCCCACGCCGGCCAACUCACCGCAGUCUGGCGCCGCCCAGGAGCCGCCCGCGCUCCCUGAACCCUAGAACUGUCUUCGACUCAGGGGCCCCGUUGGAAGACUGAGUGCCCGGGGCACGGCACAGAAGCCGCGCCCACCGCCUGCCAGUUCACAACCGCUCCGAGCGUGGGUCUCCGCCCAGCACCAGUCCUGUGAUCCGGGCCCGCCCCCUAGCGGCCGGGGAGGGAGGGGCCGGGUCCGCGGCCGGCGAACGGGGCUCGAAGGGUCCUUGUAGCCGGGAAUGCUGCUGCUGCUGCUGGGGGGAUCACAGACCAUUUCUUUCUUUCGGCCAGGCUGAGGCCCUGACGUGGAUGGGCAAACUGCAGGCCUGGGAAGGCAGCAAGCCGGGCCGUCCGUGUUCCAUCCUCCACGCACCCCCACCUAUCGUUGGUUCGCAAAGUGCAAAGCUUUCUUGUGCAUGACGCCCUGCUCUGGGGAGCGUCUGGCGCGAUCUCUGCCUGCUUACCCGGGAAAUUUGCUUUUGCCAAACCCGCUUUUUCGGGGAUCCCGCGCCCCCCUCCUCACUUGCGCUGCUCUCGGAGCCCCAGCCGGCUCCGCCCGCUUCGGCGGUUUGGAUAUUUAUUGACCUCGUCCUCCGACUCGCUGACAGGCUACAGGACCCCCAACAACCCCAAUCCACGUUUUGGAUGCACUGAGACCCCGACAUUCCUCGGUAUUUAUUGUCUGUCCCCACCUAGGACCCCCACCCCCGACCCUCGCGAAUAAAAGGCCCUCCAUCUGCCCAAAAAAAAAAAAAAAA 2GGGACAGCCAGGGACAGGCAGACAUGCAGCCAGGGCUCCAGGGCCUGGACAGGGGCUGCCAGGCCCUGUGACAGGAGGACCCCGAGCCCCCGGCCCGGGGAGGGGCCAUGGUGCUGCCUGUCCAACAUGUCAGCCGAGGUGCGGCUGAGGCGGCUCCAGCAGCUGGUGUUGGACCCGGGCUUCCUGGGGCUGGAGCCCCUGCUCGACCUUCUCCUGGGCGUCCACCAGGAGCUGGGCGCCUCCGAACUGGCCCAGGACAAGUACGUGGCCGACUUCUUGCAGUGGGCGGAGCCCAUCGUGGUGAGGCUUAAGGAGGUCCGACUGCAGAGGGACGACUUCGAGAUUCUGAAGGUGAUCGGACGCGGGGCGUUCAGCGAGGUAGCGGUAGUGAAGAUGAAGCAGACGGGCCAGGUGUAUGCCAUGAAGAUCAUGAACAAGUGGGACAUGCUGAAGAGGGGCGAGGUGUCGUGCUUCCGUGAGGAGAGGGACGUGUUGGUGAAUGGGGACCGGCGGUGGAUCACGCAGCUGCACUUCGCCUUCCAGGAUGAGAACUACCUGUACCUGGUCAUGGAGUAUUACGUGGGCGGGGACCUGCUGACACUGCUGAGCAAGUUUGGGGAGCGGAUUCCGGCCGAGAUGGCGCGCUUCUACCUGGCGGAGAUUGUCAUGGCCAUAGACUCGGUGCACCGGCUUGGCUACGUGCACAGGGACAUCAAACCCGACAACAUCCUGCUGGACCGCUGUGGCCACAUCCGCCUGGCCGACUUCGGCUCUUGCCUCAAGCUGCGGGCAGAUGGAACGGUGCGGUCGCUGGUGGCUGUGGGCACCCCAGACUACCUGUCCCCCGAGAUCCUGCAGGCUGUGGGCGGUGGGCCUGGGACAGGCAGCUACGGGCCCGAGUGUGACUGGUGGGCGCUGGGUGUAUUCGCCUAUGAAAUGUUCUAUGGGCAGACGCCCUUCUACGCGGAUUCCACGGCGGAGACCUAUGGCAAGAUCGUCCACUACAAGGAGCACCUCUCUCUGCCGCUGGUGGACGAAGGGGUCCCUGAGGAGGCUCGAGACUUCAUUCAGCGGUUGCUGUGUCCCCCGGAGACACGGCUGGGCCGGGGUGGAGCAGGCGACUUCCGGACACAUCCCUUCUUCUUUGGCCUCGACUGGGAUGGUCUCCGGGACAGCGUGCCCCCCUUUACACCGGAUUUCGAAGGUGCCACCGACACAUGCAACUUCGACUUGGUGGAGGACGGGCUCACUGCCAUGGUGAGCGGGGGCGGGGAGACACUGUCGGACAUUCGGGAAGGUGCGCCGCUAGGGGUCCACCUGCCUUUUGUGGGCUACUCCUACUCCUGCAUGGCCCUCAGGGACAGUGAGGUCCCAGGCCCCACACCCAUGGAACUGGAGGCCGAGCAGCUGCUUGAGCCACACGUGCAAGCGCCCAGCCUGGAGCCCUCGGUGUCCCCACAGGAUGAAACAGCUGAAGUGGCAGUUCCAGCGGCUGUCCCUGCGGCAGAGGCUGAGGCCGAGGUGACGCUGCGGGAGCUCCAGGAAGCCCUGGAGGAGGAGGUGCUCACCCGGCAGAGCCUGAGCCGGGAGAUGGAGGCCAUCCGCACGGACAACCAGAACUUCGCCAGUCAACUACGCGAGGCAGAGGCUCGGAACCGGGACCUAGAGGCACACGUCCGGCAGUUGCAGGAGCGGAUGGAGUUGCUGCAGGCAGAGGGAGCCACAGCUGUCACGGGGGUCCCCAGUCCCCGGGCCACGGAUCCACCUUCCCAUCUAGAUGGCCCCCCGGCCGUGGCUGUGGGCCAGUGCCCGCUGGUGGGGCCAGGCCCCAUGCACCGCCGCCACCUGCUGCUCCCUGCCAGGGUCCCUAGGCCUGGCCUAUCGGAGGCGCUUUCCCUGCUCCUGUUCGCCGUUGUUCUGUCUCGUGCCGCCGCCCUGGGCUGCAUUGGGUUGGUGGCCCACGCCGGCCAACUCACCGCAGUCUGGCGCCGCCCAGGAGCCGCCCGCGCUCCCUGAACCCUAGAACUGUCUUCGACUCCGGGGCCCCGUUGGAAGACUGAGUGCCCGGGGCACGGCACAGAAGCCGCGCCCACCGCCUGCCAGUUCACAACCGCUCCGAGCGUGGGUCUCCGCCCAGCUCCAGUCCUGUGAUCCGGGCCCGCCCCCUAGCGGCCGGGGAGGGAGGGGCCGGGUCCGCGGCCGGCGAACGGGGCUCGAAGGGUCCUUGUAGCCGGGAAUGCUGCUGCUGCUGCUGGGGGGAUCACAGACCAUUUCUUUCUUUCGGCCAGGCUGAGGCCCUGACGUGGAUGGGCAAACUGCAGGCCUGGGAAGGCAGCAAGCCGGGCCGUCCGUGUUCCAUCCUCCACGCACCCCCACCUAUCGUUGGUUCGCAAAGUGCAAAGCUUUCUUGUGCAUGACGCCCUGCUCUGGGGAGCGUCUGGCGCGAUCUCUGCCUGCUUACUCGGGAAAUUUGCUUUUGCCAAACCCGCUUUUUCGGGGAUCCCGCGCCCCCCUCCUCACUUGCGCUGCUCUCGGAGCCCCAGCCGGCUCCGCCCGCUUCGGCGGUUUGGAUAUUUAUUGACCUCGUCCUCCGACUCGCUGACAGGCUACAGGACCCCCAACAACCCCAAUCCACGUUUUGGAUGCACUGAGACCCCGACAUUCCUCGGUAUUUAUUGUCUGUCCCCACCUAGGACCCCCACCCCCGACCCUCGCGAAUAAAAGGCCCUCCAUCUGCCCAAAAAAAAAAAAAAAAAAAAA AAAAAAAA

The terms “dystrophia myotonica protein kinase” and “DMPK” as usedherein include, for example, forms of the human DMPK gene that have anucleic acid sequence that is at least 85% identical to the nucleic acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%identical to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2)and/or forms of the human DMPK gene that encode a DMPK protein havingone or more (e.g., up to 25) conservative amino acid substitutionsrelative to a wild-type DMPK protein. The terms “dystrophia myotonicaprotein kinase” and “DMPK” as used herein additionally include DMPK RNAtranscripts containing expanded CUG trinucleotide repeat regionsrelative to the length of the CUG trinucleotide repeat region of awild-type DMPK mRNA transcript. The expanded repeat region may contain,for example, 50 or more CUG trinucleotide repeats, such as from about 50to about 4,000 CUG trinucleotide repeats (e.g., about 50 CUGtrinucleotide repeats, about 60 CUG trinucleotide repeats, about 70trinucleotide repeats, 80 trinucleotide repeats, 90 trinucleotiderepeats, 100 trinucleotide repeats, 110 trinucleotide repeats, 120trinucleotide repeats, 130 trinucleotide repeats, 140 trinucleotiderepeats, 150 trinucleotide repeats, 160 trinucleotide repeats, 170trinucleotide repeats, 180 trinucleotide repeats, 190 trinucleotiderepeats, 200 trinucleotide repeats, 210 trinucleotide repeats, 220trinucleotide repeats, 230 trinucleotide repeats, 240 trinucleotiderepeats, 250 trinucleotide repeats, 260 trinucleotide repeats, 270trinucleotide repeats, 280 trinucleotide repeats, 290 trinucleotiderepeats, 300 trinucleotide repeats, 310 trinucleotide repeats, 320trinucleotide repeats, 330 trinucleotide repeats, 340 trinucleotiderepeats, 350 trinucleotide repeats, 360 trinucleotide repeats, 370trinucleotide repeats, 380 trinucleotide repeats, 390 trinucleotiderepeats, 400 trinucleotide repeats, 410 trinucleotide repeats, 420trinucleotide repeats, 430 trinucleotide repeats, 440 trinucleotiderepeats, 450 trinucleotide repeats, 460 trinucleotide repeats, 470trinucleotide repeats, 480 trinucleotide repeats, 490 trinucleotiderepeats, 500 trinucleotide repeats, 510 trinucleotide repeats, 520trinucleotide repeats, 530 trinucleotide repeats, 540 trinucleotiderepeats, 550 trinucleotide repeats, 560 trinucleotide repeats, 570trinucleotide repeats, 580 trinucleotide repeats, 590 trinucleotiderepeats, 600 trinucleotide repeats, 610 trinucleotide repeats, 620trinucleotide repeats, 630 trinucleotide repeats, 640 trinucleotiderepeats, 650 trinucleotide repeats, 660 trinucleotide repeats, 670trinucleotide repeats, 680 trinucleotide repeats, 690 trinucleotiderepeats, 700 trinucleotide repeats, 710 trinucleotide repeats, 720trinucleotide repeats, 730 trinucleotide repeats, 740 trinucleotiderepeats, 750 trinucleotide repeats, 760 trinucleotide repeats, 770trinucleotide repeats, 780 trinucleotide repeats, 790 trinucleotiderepeats, 800 trinucleotide repeats, 810 trinucleotide repeats, 820trinucleotide repeats, 830 trinucleotide repeats, 840 trinucleotiderepeats, 850 trinucleotide repeats, 860 trinucleotide repeats, 870trinucleotide repeats, 880 trinucleotide repeats, 890 trinucleotiderepeats, 900 trinucleotide repeats, 910 trinucleotide repeats, 920trinucleotide repeats, 930 trinucleotide repeats, 940 trinucleotiderepeats, 950 trinucleotide repeats, 960 trinucleotide repeats, 970trinucleotide repeats, 980 trinucleotide repeats, 990 trinucleotiderepeats, 1,000 trinucleotide repeats, 1,100 trinucleotide repeats, 1,200trinucleotide repeats, 1,300 trinucleotide repeats, 1,400 trinucleotiderepeats, 1,500 trinucleotide repeats, 1,600 trinucleotide repeats, 1,700trinucleotide repeats, 1,800 trinucleotide repeats, 1,900 trinucleotiderepeats, 2,000 trinucleotide repeats, 2,100 trinucleotide repeats, 2,200trinucleotide repeats, 2,300 trinucleotide repeats, 2,400 trinucleotiderepeats, 2,500 trinucleotide repeats, 2,600 trinucleotide repeats, 2,700trinucleotide repeats, 2,800 trinucleotide repeats, 2,900 trinucleotiderepeats, 3,000 trinucleotide repeats, 3,100 trinucleotide repeats, 3,200trinucleotide repeats, 3,300 trinucleotide repeats, 3,400 trinucleotiderepeats, 3,500 trinucleotide repeats, 3,600 trinucleotide repeats, 3,700trinucleotide repeats, 3,800 trinucleotide repeats, 3,900 trinucleotiderepeats, or 4,000 trinucleotide repeats, among others).

As used herein, the term “interfering RNA” refers to a RNA, such as ashort interfering RNA (siRNA), micro RNA (miRNA), or short hairpin RNA(shRNA) that suppresses the expression of a target RNA transcript by wayof (i) annealing to the target RNA transcript, thereby forming a nucleicacid duplex; and (ii) promoting the nuclease-mediated degradation of theRNA transcript and/or (iii) slowing, inhibiting, or preventing thetranslation of the RNA transcript, such as by sterically precluding theformation of a functional ribosome-RNA transcript complex or otherwiseattenuating formation of a functional protein product from the targetRNA transcript. Interfering RNAs as described herein may be provided toa patient, such as a human patient having myotonic dystrophy, in theform of, for example, a single- or double-stranded oligonucleotide, orin the form of a vector (e.g., a viral vector, such as anadeno-associated viral vector described herein) containing a transgeneencoding the interfering RNA. Exemplary interfering RNA platforms aredescribed, for example, in Lam et al., Molecular Therapy-Nucleic Acids4:e252 (2015); Rao et al., Advanced Drug Delivery Reviews 61:746-769(2009); and Borel et al., Molecular Therapy 22:692-701 (2014), thedisclosures of each of which are incorporated herein by reference intheir entirety.

As used herein, the “length” of a nucleic acid refers to the linear sizeof the nucleic acid as assessed by measuring the quantity of nucleotidesfrom the 5′ to the 3′ end of the nucleic acid. Exemplary molecularbiology techniques that may be used to determine the length of a nucleicacid of interest are known in the art.

As used herein, the term “myotonic dystrophy” refers to an inheritedmuscle wasting disorder characterized by the nuclear retention of RNAtranscripts encoding DMPK and containing an expanded CUG trinucleotiderepeat region in the 3′ untranslated region (UTR), such as an expandedCUG trinucleotide repeat region having from 50 to 4,000 CUG repeats.Wild-type RMPK RNA transcripts, by comparison, typically contain from 5to 37 CUG repeats in the 3′ UTR. In patients having myotonic dystrophy,the expanded CUG repeat region interacts with RNA-binding splicingfactors, such as muscleblind-like protein. This interaction causes themutant transcript to be retained in nuclear foci and leads tosequestration of RNA-binding proteins away from other pre-mRNAsubstrates, which, in turn, promotes spliceopathy of proteins involvedin modulating muscle structure and function. In type I myotonicdystrophy (DM1), skeletal muscle is often the most severely affectedtissue, but the disease also imparts toxic effects on cardiac and smoothmuscle, the ocular lens, and the brain. The cranial, distal limb, anddiaphragm muscles are preferentially affected. Manual dexterity iscompromised early, which causes several decades of severe disability.The median age at death of myotonic dystrophy patients is 55 years,which is usually caused by respiratory failure (de Die-Smulders C E, etal., Brain 121:1557-1563 (1998)).

As used herein, the term “operably linked” refers to a first molecule(e.g., a first nucleic acid) joined to a second molecule (e.g., a secondnucleic acid), wherein the molecules are so arranged that the firstmolecule affects the function of the second molecule. The two moleculesmay or may not be part of a single contiguous molecule and may or maynot be adjacent to one another. For example, a promoter is operablylinked to a transcribable polynucleotide molecule if the promotermodulates transcription of the transcribable polynucleotide molecule ofinterest in a cell. Additionally, two portions of a transcriptionregulatory element are operably linked to one another if they are joinedsuch that the transcription-activating functionality of one portion isnot adversely affected by the presence of the other portion. Twotranscription regulatory elements may be operably linked to one anotherby way of a linker nucleic acid (e.g., an intervening non-coding nucleicacid) or may be operably linked to one another with no interveningnucleotides present.

As used herein, one segment of a nucleic acid molecule is considered to“overlap with” another segment of the same nucleic acid molecule if thetwo segments share one or more constituent nucleotides. For example, twosegments of the same nucleic acid molecule are considered to “overlapwith” one another if the two segments share 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or more, constituentnucleotides. The two segments are not considered to “overlap with” oneanother if the two segments have zero constituent nucleotides in common.

“Percent (%) sequence complementarity” with respect to a referencepolynucleotide sequence is defined as the percentage of nucleic acids ina candidate sequence that are complementary to the nucleic acids in thereference polynucleotide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequencecomplementarity. A given nucleotide is considered to be “complementary”to a reference nucleotide as described herein if the two nucleotidesform canonical Watson-Crick base pairs. For the avoidance of doubt,Watson-Crick base pairs in the context of the present disclosure includeadenine-thymine, adenine-uracil, and cytosine-guanine base pairs. Aproper Watson-Crick base pair is referred to in this context as a“match,” while each unpaired nucleotide, and each incorrectly pairednucleotide, is referred to as a “mismatch.” Alignment for purposes ofdetermining percent nucleic acid sequence complementarity can beachieved in various ways that are within the capabilities of one ofskill in the art, for example, using publicly available computersoftware such as BLAST, BLAST-2, or Megalign software. Those skilled inthe art can determine appropriate parameters for aligning sequences,including any algorithms needed to achieve maximal complementarity overthe full length of the sequences being compared. As an illustration, thepercent sequence complementarity of a given nucleic acid sequence, A, toa given nucleic acid sequence, B, (which can alternatively be phrased asa given nucleic acid sequence, A that has a certain percentcomplementarity to a given nucleic acid sequence, B) is calculated asfollows:

100 multiplied by (the fraction X/Y)

where X is the number of complementary base pairs in an alignment (e.g.,as executed by computer software, such as BLAST) in that program'salignment of A and B, and where Y is the total number of nucleic acidsin B. It will be appreciated that where the length of nucleic acidsequence A is not equal to the length of nucleic acid sequence B, thepercent sequence complementarity of A to B will not equal the percentsequence complementarity of B to A. As used herein, a query nucleic acidsequence is considered to be “completely complementary” to a referencenucleic acid sequence if the query nucleic acid sequence has 100%sequence complementarity to the reference nucleic acid sequence.

“Percent (%) sequence identity” with respect to a referencepolynucleotide or polypeptide sequence is defined as the percentage ofnucleic acids or amino acids in a candidate sequence that are identicalto the nucleic acids or amino acids in the reference polynucleotide orpolypeptide sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleic acid or amino acidsequence identity can be achieved in various ways that are within thecapabilities of one of skill in the art, for example, using publiclyavailable computer software such as BLAST, BLAST-2, or Megalignsoftware. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For example, percent sequence identity values may be generated using thesequence comparison computer program BLAST. As an illustration, thepercent sequence identity of a given nucleic acid or amino acidsequence, A, to, with, or against a given nucleic acid or amino acidsequence, B, (which can alternatively be phrased as a given nucleic acidor amino acid sequence, A that has a certain percent sequence identityto, with, or against a given nucleic acid or amino acid sequence, B) iscalculated as follows:

100 multiplied by (the fraction X/Y)

where X is the number of nucleotides or amino acids scored as identicalmatches by a sequence alignment program (e.g., BLAST) in that program'salignment of A and B, and where Y is the total number of nucleic acidsin B. It will be appreciated that where the length of nucleic acid oramino acid sequence A is not equal to the length of nucleic acid oramino acid sequence B, the percent sequence identity of A to B will notequal the percent sequence identity of B to A.

As used herein, the term “pharmaceutical composition” refers to amixture containing a therapeutic agent, such as a nucleic acid or vectordescribed herein, optionally in combination with one or morepharmaceutically acceptable excipients, diluents, and/or carriers, to beadministered to a subject, such as a mammal, e.g., a human, in order toprevent, treat or control a particular disease or condition affecting orthat may affect the subject.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms, which aresuitable for contact with the tissues of a subject, such as a mammal(e.g., a human) without excessive toxicity, irritation, allergicresponse and other problem complications commensurate with a reasonablebenefit/risk ratio.

As used herein, the term “repeat region” refers to segments within agene of interest or an RNA transcript thereof containing nucleic acidrepeats, such as the poly CTG sequence in the 3″ UTR of the human DMPKgene (or the poly CUG sequence in the 3′ UTR of the RNA transcriptthereof). A repeat region is considered to be an “expanded repeatregion,” a “repeat expansion,” or the like, if the number of nucleotiderepeats in the repeat region exceeds the quantity of repeats ordinarilyfound in the repeat region of a wild-type form of the gene or RNAtranscript thereof. For example, the 3′ UTRs of wild-type human DMPKgenes typically contain from 5 to 37 CTG or CUG repeats. “Expandedrepeat regions” and “repeat expansions” in the context of the DMPK geneor an RNA transcript thereof thus refer to repeat regions containinggreater than 37 CTG or CUG repeats, such as from about 50 to about 4,000CUG trinucleotide repeats (e.g., about 50 CUG trinucleotide repeats,about 60 CUG trinucleotide repeats, about 70 trinucleotide repeats, 80trinucleotide repeats, 90 trinucleotide repeats, 100 trinucleotiderepeats, 110 trinucleotide repeats, 120 trinucleotide repeats, 130trinucleotide repeats, 140 trinucleotide repeats, 150 trinucleotiderepeats, 160 trinucleotide repeats, 170 trinucleotide repeats, 180trinucleotide repeats, 190 trinucleotide repeats, 200 trinucleotiderepeats, 210 trinucleotide repeats, 220 trinucleotide repeats, 230trinucleotide repeats, 240 trinucleotide repeats, 250 trinucleotiderepeats, 260 trinucleotide repeats, 270 trinucleotide repeats, 280trinucleotide repeats, 290 trinucleotide repeats, 300 trinucleotiderepeats, 310 trinucleotide repeats, 320 trinucleotide repeats, 330trinucleotide repeats, 340 trinucleotide repeats, 350 trinucleotiderepeats, 360 trinucleotide repeats, 370 trinucleotide repeats, 380trinucleotide repeats, 390 trinucleotide repeats, 400 trinucleotiderepeats, 410 trinucleotide repeats, 420 trinucleotide repeats, 430trinucleotide repeats, 440 trinucleotide repeats, 450 trinucleotiderepeats, 460 trinucleotide repeats, 470 trinucleotide repeats, 480trinucleotide repeats, 490 trinucleotide repeats, 500 trinucleotiderepeats, 510 trinucleotide repeats, 520 trinucleotide repeats, 530trinucleotide repeats, 540 trinucleotide repeats, 550 trinucleotiderepeats, 560 trinucleotide repeats, 570 trinucleotide repeats, 580trinucleotide repeats, 590 trinucleotide repeats, 600 trinucleotiderepeats, 610 trinucleotide repeats, 620 trinucleotide repeats, 630trinucleotide repeats, 640 trinucleotide repeats, 650 trinucleotiderepeats, 660 trinucleotide repeats, 670 trinucleotide repeats, 680trinucleotide repeats, 690 trinucleotide repeats, 700 trinucleotiderepeats, 710 trinucleotide repeats, 720 trinucleotide repeats, 730trinucleotide repeats, 740 trinucleotide repeats, 750 trinucleotiderepeats, 760 trinucleotide repeats, 770 trinucleotide repeats, 780trinucleotide repeats, 790 trinucleotide repeats, 800 trinucleotiderepeats, 810 trinucleotide repeats, 820 trinucleotide repeats, 830trinucleotide repeats, 840 trinucleotide repeats, 850 trinucleotiderepeats, 860 trinucleotide repeats, 870 trinucleotide repeats, 880trinucleotide repeats, 890 trinucleotide repeats, 900 trinucleotiderepeats, 910 trinucleotide repeats, 920 trinucleotide repeats, 930trinucleotide repeats, 940 trinucleotide repeats, 950 trinucleotiderepeats, 960 trinucleotide repeats, 970 trinucleotide repeats, 980trinucleotide repeats, 990 trinucleotide repeats, 1,000 trinucleotiderepeats, 1,100 trinucleotide repeats, 1,200 trinucleotide repeats, 1,300trinucleotide repeats, 1,400 trinucleotide repeats, 1,500 trinucleotiderepeats, 1,600 trinucleotide repeats, 1,700 trinucleotide repeats, 1,800trinucleotide repeats, 1,900 trinucleotide repeats, 2,000 trinucleotiderepeats, 2,100 trinucleotide repeats, 2,200 trinucleotide repeats, 2,300trinucleotide repeats, 2,400 trinucleotide repeats, 2,500 trinucleotiderepeats, 2,600 trinucleotide repeats, 2,700 trinucleotide repeats, 2,800trinucleotide repeats, 2,900 trinucleotide repeats, 3,000 trinucleotiderepeats, 3,100 trinucleotide repeats, 3,200 trinucleotide repeats, 3,300trinucleotide repeats, 3,400 trinucleotide repeats, 3,500 trinucleotiderepeats, 3,600 trinucleotide repeats, 3,700 trinucleotide repeats, 3,800trinucleotide repeats 3,900 trinucleotide repeats, or 4,000trinucleotide repeats, among others).

As used herein, the term “RNA dominance” refers to a pathology that isinduced by the expression and nuclear retention of RNA transcriptscontaining expanded repeat regions relative to the quantity of repeatregions, if any, contained by a wild-type form of the RNA transcript ofinterest. The toxic effects of RNA dominance are a manifestation, forexample, of the binding interaction between the expanded repeat regionsof the pathologic, mutant RNA transcripts with splicing factor proteins,which promotes the sequestration of splicing factors away from pre-mRNAtranscripts, thereby engendering spliceopathy among such substrates.Exemplary disorders associated with RNA dominance are myotonic dystrophyand amyotrophic lateral sclerosis, as described herein, among others.

As used herein, the term “sample” refers to a specimen (e.g., blood,blood component (e.g., serum or plasma), urine, saliva, amniotic fluid,cerebrospinal fluid, tissue (e.g., placental or dermal), pancreaticfluid, chorionic villus sample, or cells) isolated from a subject. Thesubject may be, for example, a patient suffering from a diseasedescribed herein, such as a heritable muscle wasting disorder (e.g.,muscular dystrophy, such as myotonic dystrophy (e.g., myotonic dystrophytype I).

As used herein, the phrases “specifically binds” and “binds” refer to abinding reaction which is determinative of the presence of a particularmolecule, such as an RNA transcript, in a heterogeneous population ofions, salts, small molecules, and/or proteins that is recognized, e.g.,by a ligand or receptor, such as an RNA-binding splicing factor protein,with particularity. A ligand (e.g., an RNA-binding protein describedherein) that specifically binds to a species (e.g., an RNA transcript)may bind to the species, e.g., with a K_(D) of less than 1 mM. Forexample, a ligand that specifically binds to a species may bind to thespecies with a K_(D) of up to 100 μM (e.g., between 1 μM and 100 μM). Aligand that does not exhibit specific binding to another molecule mayexhibit a K_(D) of greater than 1 mM (e.g., 1 μM, 100 μM, 500 μM, 1 mM,or greater) for that particular molecule or ion. A variety of assayformats may be used to determine the affinity of a ligand for a specificprotein. For example, solid-phase ELISA assays are routinely used toidentify ligands that specifically bind a target protein. See, e.g.,Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPress, New York (1988) and Harlow & Lane, Using Antibodies, A LaboratoryManual, Cold Spring Harbor Press, New York (1999), for a description ofassay formats and conditions that can be used to determine specificprotein binding.

As used herein, the term “spliceopathy” refers to a change in thesplicing pattern of an mRNA transcript that leads to the expression ofone or more alternative splice products relative to a wild-type form ofthe mRNA transcript of interest. Spliceopathy can lead to a toxic lossof function if, for example, the mRNA transcript is spliced in such away that one or more exons necessary for the activity of the encodedprotein are no longer present in the mRNA transcript upon translation.Additionally or alternatively, toxic loss of function may occur due tothe aberrant inclusion of one or more introns, for example, in a mannerthat precludes the proper folding of the encoded protein.

As used herein, the terms “subject” and “patient” refer to an organismthat receives treatment for a particular disease or condition asdescribed herein (such as a heritable muscle-wasting disorder, e.g.,myotonic dystrophy). Examples of subjects and patients include mammals,such as humans, receiving treatment for a disease or condition describedherein.

As used herein, the term “transcription regulatory element” refers to anucleic acid that controls, at least in part, the transcription of agene of interest. Transcription regulatory elements may includepromoters, enhancers, and other nucleic acids (e.g., polyadenylationsignals) that control or help to control gene transcription. Examples oftranscription regulatory elements are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185 (AcademicPress, San Diego, Calif., 1990).

As used herein, the terms “treat” or “treatment” refer to therapeutictreatment, in which the object is to prevent or slow down (lessen) anundesired physiological change or disorder, such as the progression of aheritable muscle-wasting disorder, for example, myotonic dystrophy, andparticularly, type I myotonic dystrophy. In the context of myotonicdystrophy treatment, beneficial or desired clinical results that areindicative of successful treatment include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. Treatment of a patient having myotonic dystrophy (e.g.,type I myotonic dystrophy may manifest in one or more detectablechanges, such as a decrease in the expression of DMPK RNA transcriptsthat contain expanded CUG trinucleotide repeat regions (e.g., a decreasein the expression of DMPK RNA transcripts that contain expanded CUGtrinucleotide repeat regions of 1% or more, such as a decrease of 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, relative tothe expression of DMPK RNA transcripts containing expanded CUGtrinucleotide repeat regions by the patient prior to administration of atherapeutic agent, such as a vector or nucleic acid described herein.Methods that can be used to assess RNA expression levels are known inthe art and include RNA-seq assays and polymerase chain reactiontechniques described herein. Additional clinical indications ofsuccessful treatment of a CPVT patient include alleviation ofspliceopathy, for example, of an RNA transcript that is spliced in amanner that is dependent upon muscleblind-like protein. For example,observations that signal successful treatment of a patient havingmyotonic dystrophy include a finding that the patient exhibits anincrease in corrective splicing of one or more RNA transcript substratesof muscleblind-like protein following administration of a therapeuticagent, such as a therapeutic agent described herein. For example,indicators that signal successful treatment of myotonic dystrophyinclude a determination that the patient exhibits an increase inexpression of sarcoplasmic/endoplasmic reticulum calcium ATPase 1(SERCA1) mRNA containing exon 22, such as an increase of about 1.1-foldto about 10-fold, or more (e.g., an increase in expression of SERCA1mRNA containing exon 22 by about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold,2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold,2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold,3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 4.1-fold, 4.2-fold,4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold,5-fold, 5.1-fold, 5.2-fold, 5.3-fold, 5.4-fold, 5.5-fold, 5.6-fold,5.7-fold, 5.8-fold, 5.9-fold, 6-fold, 6.1-fold, 6.2-fold, 6.3-fold,6.4-fold, 6.5-fold, 6.6-fold, 6.7-fold, 6.8-fold, 6.9-fold, 7-fold,7.1-fold, 7.2-fold, 7.3-fold, 7.4-fold, 7.5-fold, 7.6-fold, 7.7-fold,7.8-fold, 7.9-fold, 8-fold, 8.1-fold, 8.2-fold, 8.3-fold, 8.4-fold,8.5-fold, 8.6-fold, 8.7-fold, 8.8-fold, 8.9-fold, 9-fold, 9.1-fold,9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold, 9.6-fold, 9.7-fold, 9.8-fold,9.9-fold, 10-fold, or more), as assessed, for example, using an RNA orprotein detection assay described herein. Treatment of myotonicdystrophy may also manifest as a decrease in expression of chloridevoltage-gated channel 1 (CLCN1) mRNA containing exon 7a, such as adecrease of about 1% to about 100% (e.g., a decrease in expression ofCLCN1 mRNA containing exon 7a by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), as assessed, for example,using an RNA or protein detection assay described herein. Additionally,successful treatment may be signaled by a determination that the patientexhibits a decrease in expression of ZO-2 associated speckle protein(ZASP) containing exon 11, such as a decrease of about 1% to about 100%(e.g., a decrease in expression of ZASP mRNA containing exon 11 by about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%,45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%), as assessed, for example, using an RNA or protein detection assaydescribed herein. Successful treatment of myotonic dystrophy may also besignaled by a finding that, following the therapy, the patient exhibitsan increase in corrective splicing of RNA transcripts encoding insulinreceptor, ryanodine receptor 1 (RYR1), cardiac muscle troponin, and/orskeletal muscle troponin, such as an increase of about 1.1-fold to about10-fold, or more (e.g., an increase in expression of correctly splicedRNA transcripts encoding insulin receptor, RYR1, cardiac muscletroponin, and/or skeletal muscle troponin by about 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold,2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3-fold,3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold,4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold,4.8-fold, 4.9-fold, 5-fold, 5.1-fold, 5.2-fold, 5.3-fold, 5.4-fold,5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6-fold, 6.1-fold,6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold, 6.6-fold, 6.7-fold, 6.8-fold,6.9-fold, 7-fold, 7.1-fold, 7.2-fold, 7.3-fold, 7.4-fold, 7.5-fold,7.6-fold, 7.7-fold, 7.8-fold, 7.9-fold, 8-fold, 8.1-fold, 8.2-fold,8.3-fold, 8.4-fold, 8.5-fold, 8.6-fold, 8.7-fold, 8.8-fold, 8.9-fold,9-fold, 9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold, 9.6-fold,9.7-fold, 9.8-fold, 9.9-fold, 10-fold, or more), as assessed, forexample, using an RNA or protein detection assay described herein.Additional clinical indications of successful treatment of myotonicdystrophy include improvements in muscle function, such as in thecranial, distal limb, and diaphragm muscles.

As used herein, the term “vector” refers to a nucleic acid, e.g., DNA orRNA, that may function as a vehicle for the delivery of a gene ofinterest into a cell (e.g., a mammalian cell, such as a human cell),tissue, organ, or organism, such as a patient undergoing treatment for adisease or condition described herein, for purposes of expressing anencoded transgene. Exemplary vectors useful in conjunction with thecompositions and methods described herein are plasmids, DNA vectors, RNAvectors, virions, or other suitable replicon (e.g., viral vector). Avariety of vectors have been developed for the delivery ofpolynucleotides encoding exogenous proteins into a prokaryotic oreukaryotic cell. Examples of such expression vectors are disclosed in,e.g., WO 1994/11026, the disclosure of which is incorporated herein byreference. Expression vectors described herein contain a polynucleotidesequence as well as, e.g., additional sequence elements used for theexpression of proteins and/or the integration of these polynucleotidesequences into the genome of a mammalian cell. Certain vectors that canbe used for the expression of transgenes described herein includeplasmids that contain regulatory sequences, such as promoter andenhancer regions, which direct gene transcription. Other useful vectorsfor expression of transgenes contain polynucleotide sequences thatenhance the rate of translation of these genes or improve the stabilityor nuclear export of the mRNA that results from gene transcription.These sequence elements include, e.g., 5′ and 3′ untranslated regions,an internal ribosomal entry site (IRES), and polyadenylation signal sitein order to direct efficient transcription of the gene carried on theexpression vector. The expression vectors described herein may alsocontain a polynucleotide encoding a marker for selection of cells thatcontain such a vector. Examples of a suitable marker include genes thatencode resistance to antibiotics, such as ampicillin, chloramphenicol,kanamycin, or nourseothricin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the structure of human dystrophia myotonicaprotein kinase (DMPK) RNA, including the configuration of exons(represented by shaded rectangular boxes) and the site of the CUGtrinucleotide repeat region. Numerical values from 600 to 12,600 alongthe bottom of the figure indicate nucleotide position along the lengthof the DMPK RNA transcript. The diagram shows the regions within theDMPK RNA transcript to which various exemplary interfering RNAconstructs described herein anneal by way of sequence complementarity.

FIGS. 2A-2C are diagrams showing the schematics of three generations ofrAAV vectors encoding interfering RNA molecules (rAAV-RNAi vectors)targeting several genes of interest, such as those associated with RNAdominance. The rAAV plasmid pARAP4 includes the human alkalinephosphatase reporter gene (Hu Alk Phos) reporter gene, expressed fromthe Rous Sarcoma Virus (RSV) promoter, and the SV40 polyadenylationsequence, pA. The inverted terminal repeats (ITRs) originate from rAAV2,and the genomes are packaged in rAAV6 capsids. The newest vectormodification removes the RSV promoter sequence to prevent Hu Alk Phosexpression that limited rAAV-RNAi efficacy at higher doses due musclecell toxicity.

FIG. 3A is a diagram showing exemplary routes of administration ofrAAV-RNAi vectors into murine models of RNA dominance disorders, such asmyotonic dystrophy.

FIGS. 3B and 3C are diagrams comparing various characteristics ofmyotonic dystrophy type I (DM1) and the murine HSA^(LR) model of thisdisease. HSA^(LR) mice show characteristics of myotonic dystrophyresembling DM in humans. The HSA^(LR) transgene is derived frominsertion of a (CTG)₂₅₀ repeat in the 3′ UTR of the human skeletal actin(HSA) gene. When the transgene is expressed in mouse skeletal muscle,myotonic discharges are evident, splicing alterations occur in a varietyof mRNAs, and nuclear foci containing the expanded transgenic mRNA andsplicing factors are present.

FIG. 4 is a diagram showing the characteristics of HSA^(LR) micetransduced with rAAV6 HSA miR DM10, as described in Example 1, below.Human placental alkaline phosphatase (AP) staining indicates presence ofthe viral genome with active reporter gene expression. H&E staining ofcryosections from treated mice. Timepoint, 8 weeks post-injection, 4week-old HSA^(LR) mice.

FIGS. 5A and 5B are graphs quantifying HSA mRNA and expression of theHSA miRDM10 in seven individual HSA^(LR) mice transduced as described inExample 1, below. mRNA expression shown was assessed by qPCR at 8 weekspost-rAAV injection.

FIGS. 6A-6E are diagrams demonstrating that rAAV6 HSA miR DM10 systemicinjection improves splicing of Atp2a (SERCA1) and CLCN1 in the tibialisanterior (TA) muscle, as described in Example 1, below. In contrast, adifferent RNAi hairpin, miR DM4, is not as effective at reversing thesesplicing defects.

FIGS. 7A-7C are diagrams showing the structure and activity ofre-engineered rAAV-miR DM10 and -miR DM4 evaluated in vivo.Intramuscular injection of vectors without the RSV promoter to preventHu Alk Phos expression to assess the efficacy of gene silencing comparedto the previously tested Alk Phos expressing vectors. FIG. 7A shows aschematic diagram of the rAAV genome lacking the RSV promoter sequenceas the ‘new DM10’ and ‘new DM4’ labeled on the gels in FIG. 7B. FIG. 7Bshows analysis of splicing patterns for Atp2a1/Serca1 following IMinjection of ‘new DM10’ and ‘new DM4’ into the TA muscle compared to AlkPhos expressing ‘old DM10.’ L=low dose of 5×10⁹ vector genomes; H=highdose of 5×10¹⁰ vector genomes. −, no RSV promoter; +, RSV present invector genome. The high dose was chosen because it elicited some muscleregeneration as marked by the presence of central nuclei (CN) with IMinjection of Hu Alk Phos expressing vectors. However, no evidence ofmuscle turnover was observed at this high dose of rAAV DM10 lacking RSV,new DM10 and new DM4.

FIG. 8A is a diagram showing how purified plasmids expressing theDMPK-targeting miRNAs were transfected into HEK293 cells and RNA wasisolated and subjected to RT-qPCR to evaluate DMPK transcript engagementby the RISC complex and Dicer cleavage.

FIG. 8B is a graph showing the evaluation of the gene silencing activityof U6 DMPK miRNAs. Candidate therapeutic miRNA expression cassettes aand b showed reduction of the endogenous DMPK mRNA 48 hrs aftertransfection of HEK293 cells with 1.5 μg of plasmid DNA compared to aplasmid with no miRNA expression cassette. Eight biological replicateswere assayed per plasmid and control. Along the x-axis, “a” represents amiRNA containing a passenger strand having the nucleic acid sequence ofSEQ ID NO: 42 and a guide strand having the nucleic acid sequence of SEQID NO: 79; “b” represents a miRNA containing a passenger strand havingthe nucleic acid sequence of SEQ ID NO: 44 and a guide strand having thenucleic acid sequence of SEQ ID NO: 81; “c” represents a miRNAcontaining a passenger strand having the nucleic acid sequence of SEQ IDNO: 46 and a guide strand having the nucleic acid sequence of SEQ ID NO:83; “d” represents a miRNA containing a passenger strand having thenucleic acid sequence of SEQ ID NO: 47 and a guide strand having thenucleic acid sequence of SEQ ID NO: 84; and “none” represents cells nottreated with an anti-DMPK miRNA.

FIG. 9 is a graph showing the ability of various siRNA moleculesdescribed herein to downregulate DMPK expression in HEK293 cells, asdescribed in Example 4, below. Values along the y-axis representnormalized DMPK expression, and the x-axis shows the anti-DMPK siRNAmolecules tested. The first entry on the x-axis corresponds to treatmentof HEK293 cells with transfection vehicle only, and the second entry onthe x-axis represents treatment with an siRNA having a scrambled nucleicacid sequence as a negative control. siRNA molecules having a specifiedsequence identifier number are described herein, for example, in Table4, below. siRNA molecules labeled “Anti-DMPK” followed by analphanumeric identifier are commercially available from Thermo FisherScientific.

DETAILED DESCRIPTION

The compositions and methods described herein are useful for reducingthe occurrence of spliceopathy and for treating disorders associatedwith ribonucleic acid (RNA) dominance, such as myotonic dystrophy andamyotrophic lateral sclerosis, among others. The compositions describedherein include nucleic acids containing interfering RNA constructs thatsuppress the expression of RNA transcripts containing aberrantlyexpanded repeat regions. This activity provides an importantphysiological benefit, as various RNA transcripts harboring such repeatexpansions exhibit a heightened avidity for RNA splicing factors. Thisavidity manifests in sequestration of RNA splicing factors away fromother pre-mRNA substrates, thereby disrupting the proper splicing ofthese transcripts. Without being limited by mechanism, the compositionsdescribed herein may ameliorate this pathology by diminishing theexpression of RNA transcripts harboring expanded nucleotide repeats,thus releasing sequestered splicing factors so that they may properlyregulate the splicing of various other pre-mRNA transcripts. Forexample, the compositions and methods described herein may be used totreat disorders, such as myotonic dystrophy, associated with expressionof dystrophia myotonica protein kinase (DMPK) RNA transcripts containingan expanded CUG trinucleotide repeat region. Similarly, the compositionsand methods described herein may be used to treat amyotrophic lateralsclerosis, characterized by elevated expression of C9ORF72 RNAtranscripts containing GGGGCC (SEQ ID NO: 162) hexanucleotide repeats.

The interfering RNA constructs described herein may be in any of avariety of forms, such as short interfering RNA (siRNA), short hairpinRNA (shRNA), or micro RNA (miRNA). The interfering RNAs described hereinmay additionally be encoded by a vector, such as a viral vector. Forexample, described herein are adeno-associated viral (AAV) vectors, suchas pseudotyped AAV vectors (e.g., AAV2/8 and AAV2/9 vectors) containingtransgenes encoding interfering RNA constructs that attenuate theexpression of RNA transcripts harboring expanded nucleotide repeats.

The compositions and methods described herein provide, among otherbenefits, the advantageous feature of being able to selectively suppressthe expression of pathologic RNA transcripts among other RNAs thatcontain expanded nucleotide repeat regions. This property isparticularly beneficial in view of the prevalence of nucleotide repeatsin mammalian genomes, such as in the genomes of human patients. Usingthe compositions and methods described herein, the expression of RNAtranscripts that contain pathological nucleotide repeat expansions canbe diminished, while preserving the expression of important healthy RNAtranscripts and their encoded protein products.

This advantageous feature is based, in part, on the surprising discoverythat interfering RNA constructs that anneal to repeat-expanded RNAtargets at sites remote from the expanded repeat region can be used tosuppress the expression of these RNA transcripts and release splicingfactor proteins that would otherwise be sequestered by these molecules.The compositions and methods described herein can thus attenuate theexpression and nuclear retention of pathological RNA transcripts withoutthe necessity of containing complementary nucleotide repeat motifs.

The sections that follow provide a description of exemplary interferingRNA constructs that may be used in conjunction with the compositions andmethods described herein, as well as a description of vectors encodingsuch constructs and procedures that may be used to treat disordersassociated with spliceopathy, such as myotonic dystrophy and amyotrophiclateral sclerosis.

Methods of Treating RNA Dominance and Correcting Spliceopathy

Using the compositions and methods described herein, a patientexperiencing a spliceopathy and/or having a disease associated with RNAdominance, such as myotonic dystrophy, among others, can be administereda nucleic acid containing an interfering RNA construct, or a vectorencoding the same, so as to reduce the expression of RNA transcriptscontaining expanded repeat regions. Without being limited by mechanism,this activity provides the beneficial effect of releasing RNA-bindingproteins that bind with high avidity to the repeat expansion regions ofpathologic RNA transcripts. The release of such RNA-binding proteins isimportant, as the proteins sequestered by binding to repeat-expanded RNAtranscripts include splicing factors that would ordinarily be availableto modulate the proper splicing of various pre-mRNA transcripts. Inpatients having RNA dominance disorders, such as myotonic dystrophy,splicing factors such as muscleblind-like protein, which regulates thesplicing of various transcripts that encode proteins having importantroles in regulating muscle function, are sequestered from importantpre-mRNA substrates. The compositions and methods described herein maytreat RNA dominance disorders by promoting the degradation of RNAtranscripts containing expanded nucleotide repeat regions, therebyeffectuating the release of significant RNA-binding proteins from suchtranscripts.

Myotonic Dystrophy Type I

Myotonic dystrophy type I is the most common form of muscular dystrophyin adults, and occurs with an estimated frequency of 1 in 7,500 (HarperP S., Myotonic Dystrophy. London: W.B. Saunders Company; 2001). Thisdisease is an autosomal dominant disorder caused by expansion of anon-coding CTG repeat in the human DMPK1 gene. DMPK1 is a gene encodinga cytosolic serine/threonine kinase (Brook et al., Cell. 68:799-808(1992)). The expanded CTG repeat is located in the 3′ untranslatedregion (UTR) of DMPK1. This mutation leads to RNA dominance, a processin which expression of RNA containing an expanded CUG repeat (CUGexp)induces cell dysfunction (Osborne R J and Thornton C A., Human MolecularGenetics. 15:R162-R169 (2006)).

The mutant form of the DMPK mRNA, harboring large CUG repeats, are fullytranscribed and polyadenylated, but remain trapped in the nucleus (Daviset al., Proc. Natl. Acad. Sci. U.S.A 94:7388-7393 (1997)). These mutant,nuclear-retained mRNAs are one of the most important pathologicalfeatures of myotonic dystrophy type I. The DMPK gene normally has fromabout 5 to about 37 CTG repeats in the 3′ UTR. In myotonic dystrophytype I, this number is significantly expanded, and may be in the range,for example, of from 50 to greater than 4,000 repeats. The CUGexp tractin the ensuing RNA transcript interacts with RNA-binding splicing factorproteins, including muscleblind-like protein. The enhanced avidityengendered by the expanded CUG repeat region causes the mutanttranscript to retain such splicing factor proteins in nuclear foci. Thetoxicity of this mutant RNA stems from the sequestration of RNA-bindingsplicing factor proteins away from other pre-mRNA substrates, includingthose that encode proteins that have important roles in regulatingmuscle function.

In myotonic dystrophy type I, skeletal muscle is the most severelyaffected tissue, but the disease also has important effects on cardiacand smooth muscle, ocular lens, and the brain. Among muscle tissue, thecranial, distal limb, and diaphragm muscles are often preferentiallyaffected. Manual dexterity is compromised early, which causes severaldecades of severe disability. The median age at death is 55 years, whichusually from respiratory failure (de Die-Smulders C E, et al., Brain121(Pt 8):1557-1563 (1998)). Symptoms of myotonic dystrophy include,without limitation, myotonia, muscle stiffness, disabling distalweakness, weakness in the face and jaw muscles, difficulty inswallowing, drooping of the eyelids (ptosis), weakness of neck muscles,weakness in arm and leg muscles, persistent muscle pain, hypersomnia,muscle wasting, dysphagia, respiratory insufficiency, irregularheartbeat, heart muscle damage, apathy, insulin resistance, andcataracts. In children, symptoms may also include developmental delays,learning problems, language and speech difficulties, and personalitydevelopment challenges.

Pathogenic DMPK Transcripts

Myotonic dystrophy patients that may be treated using the compositionsand methods described herein include patients, such as human patient,having myotonic dystrophy type I, and that express a DMPK RNA transcriptharboring a CUG repeat expansion. Exemplary DMPK RNA transcripts thatmay be expressed by a patient undergoing treatment with the compositionsand methods described herein are set forth in GenBank Accession Nos.NM_001081560.1, NT_011109.15 (from nucleotides 18540696 to Ser. No.18/555,106), NT_039413.7 (from nucleotides 16666001 to Ser. No.16/681,000), NM_032418.1, AI007148.1, AI304033.1, BC024150.1,BC056615.1, BC075715.1, BU519245.1, CB247909.1, CX208906.1, CX732022.1,560315.1, 560316.1, NM_001081562.1, and NM_001100.3.

Suppression of Pathologic DMPK RNA Expression

Using the compositions and methods described herein, a patient, such asa patient suffering from myotonic dystrophy (e.g., myotonic dystrophytype I) may be administered a vector encoding, or a compositioncontaining, an interfering RNA that anneals to and suppresses theexpression of pathologic DMPK mRNA transcripts. The compositions andmethods described herein may selectively attenuate the expression ofDMPK mRNA transcripts containing expanded CUG repeats, such as DMPK mRNAtranscripts containing from about 50 to about 4,000, or more, CUGrepeats. For example, the interfering RNA molecules described herein mayactivate ribonucleases, such as nuclear ribonucleases, that specificallydigest nuclear-retained DMPK transcripts harboring CUG repeatexpansions. The decrease in mutant DMPK mRNA expression may be adecrease of, for example, about 1% or more, such as a decrease of 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, relative tothe expression of DMPK mRNA transcripts containing expanded CUGtrinucleotide repeat regions by the patient prior to administration of atherapeutic agent described herein, such as a vector or nucleic aciddescribed herein. Methods that can be used to assess RNA expressionlevels are known in the art and include RNA-seq assays and polymerasechain reaction techniques described herein.

Correction of Spliceopathy

In some embodiments, the compositions and methods described herein canbe used to correct one or more spliceopathies in a patient, such as apatient suffering from myotonic dystrophy (e.g., myotonic dystrophy typeI). Without being limited by mechanism, the ability of the interferingRNA molecules described herein to anneal to, and suppresses theexpression of, pathologic DMPK transcripts (e.g., DMPK transcriptscontaining expanded CUG repeat regions) can release splicing factors,such as muscleblind-like protein, that would otherwise be sequestered byway of binding to the CUG repeats. This release of splicing factors may,in turn, effectuate corrective splicing of one or more RNA transcriptsubstrates of these splicing factors. For example, upon administrationof the vector or composition to a patient suffering from myotonicdystrophy, the patient may exhibit an increase in expression ofsarcoplasmic/endoplasmic reticulum calcium ATPase 1 (SERCA1) mRNAcontaining exon 22, for example, in the tibialis anterior,gastrocnemius, and/or quadriceps muscles. The increase in expression ofSERCA1 mRNA transcripts containing exon 22 may be an increase of, forexample, about 1.1-fold to about 10-fold, or more (e.g., an increase inexpression of SERCA1 mRNA containing exon 22 by about 1.1-fold,1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold,2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold,3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold,4-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5-fold, 4.6-fold,4.7-fold, 4.8-fold, 4.9-fold, 5-fold, 5.1-fold, 5.2-fold, 5.3-fold,5.4-fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold, 5.9-fold, 6-fold,6.1-fold, 6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold, 6.6-fold, 6.7-fold,6.8-fold, 6.9-fold, 7-fold, 7.1-fold, 7.2-fold, 7.3-fold, 7.4-fold,7.5-fold, 7.6-fold, 7.7-fold, 7.8-fold, 7.9-fold, 8-fold, 8.1-fold,8.2-fold, 8.3-fold, 8.4-fold, 8.5-fold, 8.6-fold, 8.7-fold, 8.8-fold,8.9-fold, 9-fold, 9.1-fold, 9.2-fold, 9.3-fold, 9.4-fold, 9.5-fold,9.6-fold, 9.7-fold, 9.8-fold, 9.9-fold, 10-fold, or more), as assessed,for example, using an RNA or protein detection assay described herein.

In some embodiments, upon administration of a vector or compositiondescribed herein to a patient suffering from myotonic dystrophy, thepatient may exhibit a decrease in expression of chloride voltage-gatedchannel 1 (CLCN1) mRNA containing exon 7a, for example, in the tibialisanterior, gastrocnemius, and/or quadriceps muscles. The decrease inexpression of CLCN1 mRNA transcripts containing exon 7a may be adecrease of, for example, about 1% to about 100% (e.g., a decrease inexpression of CLCN1 mRNA containing exon 7a by about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), as assessed, forexample, using an RNA or protein detection assay described herein.

Additionally or alternatively, upon administration of a vector orcomposition described herein to a patient suffering from myotonicdystrophy, the patient may exhibit a decrease in expression of ZO-2associated speckle protein (ZASP) containing exon 11, for example, inthe tibialis anterior, gastrocnemius, and/or quadriceps muscles. Thedecrease in expression of ZASP mRNA transcripts containing exon 11 maybe a decrease of, for example, about 1% to about 100% (e.g., a decreasein expression of ZASP mRNA containing exon 11 by about 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), as assessed,for example, using an RNA or protein detection assay described herein.

Additionally or alternatively, upon administration of a vector orcomposition described herein to a patient suffering from myotonicdystrophy, the patient may exhibit an increase in corrective splicing ofRNA transcripts encoding insulin receptor, ryanodine receptor 1 (RYR1),cardiac muscle troponin, and/or skeletal muscle troponin, such as anincrease of about 1.1-fold to about 10-fold, or more (e.g., an increasein expression of correctly spliced RNA transcripts encoding insulinreceptor, RYR1, cardiac muscle troponin, and/or skeletal muscle troponinby about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold,2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold,3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold,3.8-fold, 3.9-fold, 4-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold,4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5-fold, 5.1-fold,5.2-fold, 5.3-fold, 5.4-fold, 5.5-fold, 5.6-fold, 5.7-fold, 5.8-fold,5.9-fold, 6-fold, 6.1-fold, 6.2-fold, 6.3-fold, 6.4-fold, 6.5-fold,6.6-fold, 6.7-fold, 6.8-fold, 6.9-fold, 7-fold, 7.1-fold, 7.2-fold,7.3-fold, 7.4-fold, 7.5-fold, 7.6-fold, 7.7-fold, 7.8-fold, 7.9-fold,8-fold, 8.1-fold, 8.2-fold, 8.3-fold, 8.4-fold, 8.5-fold, 8.6-fold,8.7-fold, 8.8-fold, 8.9-fold, 9-fold, 9.1-fold, 9.2-fold, 9.3-fold,9.4-fold, 9.5-fold, 9.6-fold, 9.7-fold, 9.8-fold, 9.9-fold, 10-fold, ormore), as assessed, for example, using an RNA or protein detection assaydescribed herein.

Improvement in Muscle Function

The beneficial treatment effects of the compositions and methodsdescribed herein, such as the ability of the interfering RNA moleculesdescribed herein, and the vectors encoding the same, to (i) suppresspathologic DMPK RNA expression and/or (ii) restore correct splicing ofproteins involved in regulating muscle function may manifest clinicallyin a variety of ways. For example, patients having myotonic dystrophy,such as myotonic dystrophy type I, may exhibit an improvement incranial, distal limb, and/or diaphragmatic muscle function. Theimprovement in muscle function may be observed, for example, as anincrease in muscle mass, frequency of muscle contractions, and/ormagnitude of muscle contractions. For example, using the compositionsand methods described herein, a patient suffering from myotonicdystrophy (e.g., myotonic dystrophy type I) may exhibit an increase incranial, distal limb, and/or diaphragmatic muscle mass, frequency ofmuscle contractions, and/or magnitude of muscle contractions. Theincrease in muscle mass, frequency of muscle contractions, and/ormagnitude of muscle contractions may be, for example, an increase of 1%or more, such as an increase of from 1% to 25%, from 1% to 50%, from 1%to 75%, from 1% to 100%, from 1% to 500%, from 1% to 1,000%, or more,such as an increase in muscle mass, frequency of muscle contractions,and/or magnitude of muscle contractions of about 1%, 5%, 10%, 15%, 20%,25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 80%, 900%,1,000%, or more.

Particularly, in patients having myotonic dystrophy (e.g., myotonicdystrophy type I), the beneficial therapeutic effects of the interferingRNA molecules described herein, and of the vectors encoding the same,may manifest as a reduction in myotonia. Thus, using the compositionsand methods described herein, a patient having myotonic dystrophy (e.g.,myotonic dystrophy type I) may be administered an interfering RNAmolecule or vector encoding the same so as to facilitate and/oraccelerate muscle relaxation. For example, the compositions and methodsdescribed herein may be used to accelerate muscle relaxation bysuppressing the onset of spontaneous action potentials caused byfluctuations in chloride ion concentration. Without being limited bymechanism, this beneficial activity may be caused by the restoration ofcorrect splicing of CLCN1 mRNA, for example, such that the expression ofCLCN1 mRNA containing exon 7a in the patient is reduced. As CLCN1channel protein regulates chloride ion concentration, correcting thesplicing pattern of CLCN1 mRNA transcripts may engender a reduction inthe onset of spontaneous action potentials and an improvement in musclerelaxation speed, thereby ameliorating myotonia.

Suppression of myotonia can be evaluated using a variety of techniquesknown in the art, for example, by way of electromyography. Particularly,electromyography on the left and right quadriceps, left and rightgastrocnemius muscles, left and right tibialis anterior muscles, and/orlumbar paraspinals muscles can be performed to assess the effects of thecompositions and methods described herein on myotonia in a patient, suchas a human patient having myotonic dystrophy. Electromyography protocolshave been described, for example, in Kanadia et al., Science302:1978-1980 (2003)). For example, electromyography may be performedusing 30-gauge concentric needle electrodes and a minimum of 10 needleinsertions for each muscle. In this way, an average myotonia grade maybe determined for a subject, such as a human patient or a model organism(e.g., a murine model of muscular dystrophy described herein). Thisgrade can then be compared to the average myotonia grade of the patientor model organism determined prior to administration of a therapeuticagent described herein (e.g., an interfering RNA molecule or a vectorencoding the same). A finding that the average myotonia grade hasdecreased following administration of the therapeutic agent can serve asan indication of successful treatment of myotonic dystrophy and as anindicator of successful amelioration of the symptom of myotonia.

Amelioration of Additional Myotonic Dystrophy Symptoms

Using the compositions and methods described herein, a patient havingmyotonic dystrophy, such as myotonic dystrophy type 1, may beadministered an interfering RNA molecule or vector encoding the same soas to attenuate or altogether eliminate one or more symptoms of myotonicdystrophy. Aside from the myotonia described above, symptoms of myotonicdystrophy include, without limitation, muscle stiffness, disablingdistal weakness, weakness in the face and jaw muscles, difficulty inswallowing, ptosis, weakness of neck muscles, weakness in arm and legmuscles, persistent muscle pain, hypersomnia, muscle wasting, dysphagia,respiratory insufficiency, irregular heartbeat, heart muscle damage,apathy, insulin resistance, and cataracts. In children, symptoms mayalso include developmental delays, learning problems, language andspeech difficulties, and personality development challenges. Thecompositions and methods described herein may be used to alleviate oneor more, or all, of the foregoing symptoms.

Duration of Therapeutic Effects

The compositions and methods described herein provide beneficialclinical effects that may last for extended periods of time. Forexample, using one or more of the interfering RNA molecules describedherein, and/or vector(s) encoding the same, a patient having myotonicdystrophy (e.g., myotonic dystrophy type I) may exhibit (i) a reductionin pathologic DMPK RNA expression (e.g., a reduction in expression ofDMPK RNA harboring from about 50 to about 4,000 CUG repeats, or more),(ii) an improvement in muscle function (such as an improvement in musclemass and/or muscle activity, e.g., in the cranial, distal limb, anddiaphragm muscle) and/or (iii) alleviation of one or more symptoms ofmyotonic dystrophy, for a period of one or more days, weeks, months, oryears. For example, using the compositions and methods described herein,the beneficial therapeutic effects described herein may be achieved fora period of at least 30 days, at least 35 days, at least 40 days, atleast 45 days, at least 50 days, at least 55 days, at least 60 days, atleast 65 days, at least 70 days, at least 75 days, at least 76 days, atleast 77 days, at least 78 days, at least 79 days, at least 80 days, atleast 85 days, at least 90 days, at least 95 days, at least 100 days, atleast 105 days, at least 110 days, at least 115 days, at least 120 days,or at least 1 year.

Murine Models of Myotonic Dystrophy

To examine the therapeutic effects of an interfering RNA moleculedescribed herein, or those of a vector encoding the same, an appropriatemouse model may be utilized. For example, the HSA (human skeletal actin)^(LR) (long repeat) mouse model is an established model for myotonicdystrophy type 1 (see, e.g., Mankodi et al., Science 289:1769 (2000),the disclosure of which is incorporated herein by reference as itpertains to the HSA^(LR) mouse. These mice carry a human skeletal actin(hACTA1) transgene containing an expanded CTG region. Particularly, thehACTA1 transgene in HSA^(LR) mice contains 220 CTG repeats inserted inthe 3′ UTR of the gene. Upon transcription, the hACTA1-CUGexp RNAtranscript accumulates in nuclear foci in skeletal muscles and resultsin myotonia similar to that observed in human myotonic dystrophy type 1,for example, due to the binding of CUG repeat expansions to splicingfactors and the sequestration of these splicing factors from pre-mRNAtranscripts encoding genes that play an important role in regulatingmuscle function (see, e.g., Mankodi et al., Mol. Cell 10:35 (2002), andLin et al., Hum. Mol. Genet. 15:2087 (2006), the disclosures of each ofwhich are incorporated herein by reference as they pertain to theHSA^(LR) mouse). Thus, amelioration of myotonic dystrophy type Isymptoms in the HSA^(LR) mouse by suppression of the expression ofhACTA1 RNA transcripts harboring CUG expanded repeat regions may be apredictor of amelioration of similar symptoms in human patients bysuppression of the expression of pathologic DMPK RNA transcripts.HSA^(LR) myotonic dystrophy type I mice can be generated using methodsknown in the art, for example, by insertion into the genome of FVB/Nmice of a hACTA1 transgene with 250 CUG repeats in the 3′ UTR of humanskeletal actin. The transgene is subsequently expressed in the mice as aCUG repeat expansion in hACTA1 RNA. This repeat-expanded RNA is retainedin the nucleus, forming nuclear inclusions similar to those observed inhuman tissue samples of patients with myotonic dystrophy.

As described above, in the HSA^(LR) mouse model, the accumulation ofexpanded CUG RNA in the nucleus leads to the sequestration ofpoly(CUG)-binding splicing factor proteins, such as muscleblind-likeprotein (Miller et al., EMBO J. 19:4439 (2000)). This splicing factor,which controls alternative splicing of the SERCA1 gene, is thussequestered in expanded CUG foci in HSA^(LR) mice. This sequestrationtriggers dysregulation of the alternative splicing of the SERCA1 gene.To evaluate the therapeutic effect of the interfering RNA moleculesdescribed herein, and/or of vectors encoding the same, thesecompositions may be designed so as to anneal to a region of the hACTA1RNA transcript, for example, at a site distal from the CUG repeatexpansion. This may be accomplished, for example, by designing aninterfering RNA molecule that is at least 85% complementary (e.g., atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% complementary)to a segment of hACTA1 RNA that does not overlap with the CUG repeatexpansion region. The suppression of repeat-expanded hACTA1 RNA, and theconcomitant increase in correctly spliced SERCA1 mRNA (and, thus,functional SERCA1 protein), can then be assessed using RNA and proteindetection methods known in the art and described herein. For example, tomonitor an increase in the expression of correctly spliced SERCA1 mRNAtranscripts (e.g., SERCA1 mRNA transcripts containing exon 22, followingadministration of an interfering RNA molecule designed to anneal to, andsuppress the expression of, pathologic hACTA1 RNA, total RNA may bepurified from the HSA^(LR) mouse at one or more, or all, of the tibialisanterior, gastrocnemius, and quadriceps muscle using the RNeasy LipidTissue Mini Kit (Qiagen®), according to the manufacturer's instructions.RT-PCR may be performed with, for example, the SuperScript III One-StepRT-PCR System and Platinum Taq Polymerase (Invitrogen®), usinggene-specific primers for cDNA synthesis and PCR amplification. Theforward and reverse primers for SERCA1 have been described, for example,in Bennett and Swayze, Annu Rev. Pharmacol. 50:259-293 (2010)). PCRproducts may be separated on agarose gels, stained with SybrGreen INucleic Acid Gel Stain (Invitrogen®), and imaged using a FujifilmLAS-3000 Intelligent Dark Box. Restoration of correct splicing of theSERCA1 gene by an interfering RNA molecule, or vector encoding the same,for example, in the tibialis anterior, gastrocnemius, and/or quadricepsmuscles of the HSA^(LR) mouse, may be a predictor of the therapeuticefficacy of an interfering RNA molecule, or vector encoding the same,that anneals to a similar site on human DMPK RNA.

Additional murine models of myotonic dystrophy include LC15 mice, LineA, which are transgenic mice containing the entire human DMPK 3′UTR(developed by Wheeler et al, University of Rochester). These mice arethe second generation of mice backcrossed to an FVB background. The DMPKtransgene is expressed in these mice as a CUG repeat in the DMPK RNAtranscript, which is retained in the nucleus, thereby forming nuclearinclusions similar to those observed in human tissue samples of patientswith myotonic dystrophy. LC15 mice may express DMPK RNA transcriptscontaining from about 350 to about 400 CUG repeats. These mice displayearly signs of myotonic dystrophy type I and do not display any myotoniain their muscle tissues.

Yet another murine model of myotonic dystrophy that may be used toassess the therapeutic efficacy of an interfering RNA molecule describedherein or a vector encoding the same is the DMSXL model. DMSXL mice aregenerated by way of successive breeding of mice having a high level ofCTG repeat instability, and, as a result, DMSXL mice express DMPK RNAtranscripts containing >1,000 CUG trinucleotide repeats in the 3′ UTR.DMSXL mice and methods for producing the same are described in detail,for example, in Gomes-Pereira et al., PLoS Genet. 3:e52 (2007), andHuguet et al., A, PLoS Genet. 8:e1003043 (2012), the disclosures of eachof which are incorporated herein by reference in their entirety.

Additional Disorders Characterized by Nuclear Retention ofRepeat-Expanded RNA

In addition to myotonic dystrophy type I, disorders characterized by theexpression and nuclear retention of RNA transcripts harboring expandedrepeat regions and that may be treated using the compositions andmethods described herein include myotonic dystrophy type II andamyotrophic lateral sclerosis, among others. In the case of myotonicdystrophy type II, patients may express a mutant version of the cellularnucleic acid binding protein (CNBP) gene (also known as the zinc fingerprotein 9 (ZNF9) gene) harboring a CCUG (SEQ ID NO: 164) repeatexpansion. In cases of patients having amyotrophic lateral sclerosis,patients may express mutant versions of C9ORF72 harboring expandedGGGGCC (SEQ ID NO: 162) repeats. The nucleic acid sequence of severalisoforms of C9ORF72 mRNA are shown in Table 3, below. In all instances,patients having these disorders may be treated by administration ofinterfering RNA molecules (or vectors encoding the same) that anneal toand suppress the expression of the mutant RNA transcript, therebyreleasing RNA-binding proteins that would ordinarily bind othersubstrates but are instead sequestered by virtue of high-avidity bindingto the repeat expansion regions in the mutant transcripts expressed bysuch patients. Methods for monitoring the reduction in the expression ofnuclear-retained RNA transcripts, such as pathologic CNBP and C9ORF72transcripts harboring CCUG (SEQ ID NO: 164) and GGGGCC (SEQ ID NO: 162)repeats, respectively, include various molecular biology techniquesknown in the art and described herein.

TABLE 3 Nucleic acid sequences of exemplary isoforms of C9ORF72 mRNASEQ ID NCBI Reference NO. Sequence No. Nucleic Acid Sequence 163NM_001256054.2 ACGUAACCUACGGUGUCCCGCUAGGAAAGAGAGGUGCGUCAAACAGCGACAAGUUCCGCCCACGUAAAAGAUGACGCUUGGUGUGUCAGCCGUCCCUGCUGCCCGGUUGCUUCUCUUUUGGGGGCGGGGUCUAGCAAGAGCAGGUGUGGGUUUAGGAGAUAUCUCCGGAGCAUUUGGAUAAUGUGACAGUUGGAAUGCAGUGAUGUCGACUCUUUGCCCACCGCCAUCUCCAGCUGUUGCCAAGACAGAGAUUGCUUUAAGUGGCAAAUCACCUUUAUUAGCAGCUACUUUUGCUUACUGGGACAAUAUUCUUGGUCCUAGAGUAAGGCACAUUUGGGCUCCAAAGACAGAACAGGUACUUCUCAGUGAUGGAGAAAUAACUUUUCUUGCCAACCACACUCUAAAUGGAGAAAUCCUUCGAAAUGCAGAGAGUGGUGCUAUAGAUGUAAAGUUUUUUGUCUUGUCUGAAAAGGGAGUGAUUAUUGUUUCAUUAAUCUUUGAUGGAAACUGGAAUGGGGAUCGCAGCACAUAUGGACUAUCAAUUAUACUUCCACAGACAGAACUUAGUUUCUACCUCCCACUUCAUAGAGUGUGUGUUGAUAGAUUAACACAUAUAAUCCGGAAAGGAAGAAUAUGGAUGCAUAAGGAAAGACAAGAAAAUGUCCAGAAGAUUAUCUUAGAAGGCACAGAGAGAAUGGAAGAUCAGGGUCAGAGUAUUAUUCCAAUGCUUACUGGAGAAGUGAUUCCUGUAAUGGAACUGCUUUCAUCUAUGAAAUCACACAGUGUUCCUGAAGAAAUAGAUAUAGCUGAUACAGUACUCAAUGAUGAUGAUAUUGGUGACAGCUGUCAUGAAGGCUUUCUUCUCAAUGCCAUCAGCUCACACUUGCAAACCUGUGGCUGUUCCGUUGUAGUAGGUAGCAGUGCAGAGAAAGUAAAUAAGAUAGUCAGAACAUUAUGCCUUUUUCUGACUCCAGCAGAGAGAAAAUGCUCCAGGUUAUGUGAAGCAGAAUCAUCAUUUAAAUAUGAGUCAGGGCUCUUUGUACAAGGCCUGCUAAAGGAUUCAACUGGAAGCUUUGUGCUGCCUUUCCGGCAAGUCAUGUAUGCUCCAUAUCCCACCACACACAUAGAUGUGGAUGUCAAUACUGUGAAGCAGAUGCCACCCUGUCAUGAACAUAUUUAUAAUCAGCGUAGAUACAUGAGAUCCGAGCUGACAGCCUUCUGGAGAGCCACUUCAGAAGAAGACAUGGCUCAGGAUACGAUCAUCUACACUGACGAAAGCUUUACUCCUGAUUUGAAUAUUUUUCAAGAUGUCUUACACAGAGACACUCUAGUGAAAGCCUUCCUGGAUCAGGUCUUUCAGCUGAAACCUGGCUUAUCUCUCAGAAGUACUUUCCUUGCACAGUUUCUACUUGUCCUUCACAGAAAAGCCUUGACACUAAUAAAAUAUAUAGAAGACGAUACGCAGAAGGGAAAAAAGCCCUUUAAAUCUCUUCGGAACCUGAAGAUAGACCUUGAUUUAACAGCAGAGGGCGAUCUUAACAUAAUAAUGGCUCUGGCUGAGAAAAUUAAACCAGGCCUACACUCUUUUAUCUUUGGAAGACCUUUCUACACUAGUGUGCAAGAACGAGAUGUUCUAAUGACUUUUUAAAUGUGUAACUUAAUAAGCCUAUUCCAUCACAAUCAUGAUCGCUGGUAAAGUAGCUCAGUGGUGUGGGGAAACGUUCCCCUGGAUCAUACUCCAGAAUUCUGCUCUCAGCAAUUGCAGUUAAGUAAGUUACACUACAGUUCUCACAAGAGCCUGUGAGGGGAUGUCAGGUGCAUCAUUACAUUGGGUGUCUCUUUUCCUAGAUUUAUGCUUUUGGGAUACAGACCUAUGUUUACAAUAUAAUAAAUAUUAUUGCUAUCUUUUAAAGAUAUAAUAAUAGGAUGUAAACUUGACCACAACUACUGUUUUUUUGAAAUACAUGAUUCAUGGUUUACAUGUGUCAAGGUGAAAUCUGAGUUGGCUUUUACAGAUAGUUGACUUUCUAUCUUUUGGCAUUCUUUGGUGUGUAGAAUUACUGUAAUACUUCUGCAAUCAACUGAAAACUAGAGCCUUUAAAUGAUUUCAAUUCCACAGAAAGAAAGUGAGCUUGAACAUAGGAUGAGCUUUAGAAAGAAAAUUGAUCAAGCAGAUGUUUAAUUGGAAUUGAUUAUUAGAUCCUACUUUGUGGAUUUAGUCCCUGGGAUUCAGUCUGUAGAAAUGUCUAAUAGUUCUCUAUAGUCCUUGUUCCUGGUGAACCACAGUUAGGGUGUUUUGUUUAUUUUAUUGUUCUUGCUAUUGUUGAUAUUCUAUGUAGUUGAGCUCUGUAAAAGGAAAUUGUAUUUUAUGUUUUAGUAAUUGUUGCCAACUUUUUAAAUUAAUUUUCAUUAUUUUUGAGCCAAAUUGAAAUGUGCACCUCCUGUGCCUUUUUUCUCCUUAGAAAAUCUAAUUACUUGGAACAAGUUCAGAUUUCACUGGUCAGUCAUUUUCAUCUUGUUUUCUUCUUGCUAAGUCUUACCAUGUACCUGCUUUGGCAAUCAUUGCAACUCUGAGAUUAUAAAAUGCCUUAGAGAAUAUACUAACUAAUAAGAUCUUUUUUUCAGAAACAGAAAAUAGUUCCUUGAGUACUUCCUUCUUGCAUUUCUGCCUAUGUUUUUGAAGUUGUUGCUGUUUGCCUGCAAUAGGCUAUAAGGAAUAGCAGGAGAAAUUUUACUGAAGUGCUGUUUUCCUAGGUGCUACUUUGGCAGAGCUAAGUUAUCUUUUGUUUUCUUAAUGCGUUUGGACCAUUUUGCUGGCUAUAAAAUAACUGAUUAAUAUAAUUCUAACACAAUGUUGACAUUGUAGUUACACAAACACAAAUAAAUAUUUUAUUUAAAAUUCUGGAAGUAAUAUAAAAGGGAAAAUAUAUUUAUAAGAAAGGGAUAAAGGUAAUAGAGCCCUUCUGCCCCCCACCCACCAAAUUUACACAACAAAAUGACAUGUUCGAAUGUGAAAGGUCAUAAUAGCUUUCCCAUCAUGAAUCAGAAAGAUGUGGACAGCUUGAUGUUUUAGACAACCACUGAACUAGAUGACUGUUGUACUGUAGCUCAGUCAUUUAAAAAAUAUAUAAAUACUACCUUGUAGUGUCCCAUACUGUGUUUUUUACAUGGUAGAUUCUUAUUUAAGUGCUAACUGGUUAUUUUCUUUGGCUGGUUUAUUGUACUGUUAUACAGAAUGUAAGUUGUACAGUGAAAUAAGUUAUUAAAGCAUGUGUAAACAUUGUUAUAUAUCUUUUCUCCUAAAUGGAGAAUUUUGAAUAAAAUAUAUUUGAAAUUU UAAAAAAAAAAAAAAAAAA 165NM_018325.4 GGGCGGGGCUGCGGUUGCGGUGCCUGCGCCCGCGGCGGCGGAGGCGCAGGCGGUGGCGAGUGGAUAUCUCCGGAGCAUUUGGAUAAUGUGACAGUUGGAAUGCAGUGAUGUCGACUCUUUGCCCACCGCCAUCUCCAGCUGUUGCCAAGACAGAGAUUGCUUUAAGUGGCAAAUCACCUUUAUUAGCAGCUACUUUUGCUUACUGGGACAAUAUUCUUGGUCCUAGAGUAAGGCACAUUUGGGCUCCAAAGACAGAACAGGUACUUCUCAGUGAUGGAGAAAUAACUUUUCUUGCCAACCACACUCUAAAUGGAGAAAUCCUUCGAAAUGCAGAGAGUGGUGCUAUAGAUGUAAAGUUUUUUGUCUUGUCUGAAAAGGGAGUGAUUAUUGUUUCAUUAAUCUUUGAUGGAAACUGGAAUGGGGAUCGCAGCACAUAUGGACUAUCAAUUAUACUUCCACAGACAGAACUUAGUUUCUACCUCCCACUUCAUAGAGUGUGUGUUGAUAGAUUAACACAUAUAAUCCGGAAAGGAAGAAUAUGGAUGCAUAAGGAAAGACAAGAAAAUGUCCAGAAGAUUAUCUUAGAAGGCACAGAGAGAAUGGAAGAUCAGGGUCAGAGUAUUAUUCCAAUGCUUACUGGAGAAGUGAUUCCUGUAAUGGAACUGCUUUCAUCUAUGAAAUCACACAGUGUUCCUGAAGAAAUAGAUAUAGCUGAUACAGUACUCAAUGAUGAUGAUAUUGGUGACAGCUGUCAUGAAGGCUUUCUUCUCAAUGCCAUCAGCUCACACUUGCAAACCUGUGGCUGUUCCGUUGUAGUAGGUAGCAGUGCAGAGAAAGUAAAUAAGAUAGUCAGAACAUUAUGCCUUUUUCUGACUCCAGCAGAGAGAAAAUGCUCCAGGUUAUGUGAAGCAGAAUCAUCAUUUAAAUAUGAGUCAGGGCUCUUUGUACAAGGCCUGCUAAAGGAUUCAACUGGAAGCUUUGUGCUGCCUUUCCGGCAAGUCAUGUAUGCUCCAUAUCCCACCACACACAUAGAUGUGGAUGUCAAUACUGUGAAGCAGAUGCCACCCUGUCAUGAACAUAUUUAUAAUCAGCGUAGAUACAUGAGAUCCGAGCUGACAGCCUUCUGGAGAGCCACUUCAGAAGAAGACAUGGCUCAGGAUACGAUCAUCUACACUGACGAAAGCUUUACUCCUGAUUUGAAUAUUUUUCAAGAUGUCUUACACAGAGACACUCUAGUGAAAGCCUUCCUGGAUCAGGUCUUUCAGCUGAAACCUGGCUUAUCUCUCAGAAGUACUUUCCUUGCACAGUUUCUACUUGUCCUUCACAGAAAAGCCUUGACACUAAUAAAAUAUAUAGAAGACGAUACGCAGAAGGGAAAAAAGCCCUUUAAAUCUCUUCGGAACCUGAAGAUAGACCUUGAUUUAACAGCAGAGGGCGAUCUUAACAUAAUAAUGGCUCUGGCUGAGAAAAUUAAACCAGGCCUACACUCUUUUAUCUUUGGAAGACCUUUCUACACUAGUGUGCAAGAACGAGAUGUUCUAAUGACUUUUUAAAUGUGUAACUUAAUAAGCCUAUUCCAUCACAAUCAUGAUCGCUGGUAAAGUAGCUCAGUGGUGUGGGGAAACGUUCCCCUGGAUCAUACUCCAGAAUUCUGCUCUCAGCAAUUGCAGUUAAGUAAGUUACACUACAGUUCUCACAAGAGCCUGUGAGGGGAUGUCAGGUGCAUCAUUACAUUGGGUGUCUCUUUUCCUAGAUUUAUGCUUUUGGGAUACAGACCUAUGUUUACAAUAUAAUAAAUAUUAUUGCUAUCUUUUAAAGAUAUAAUAAUAGGAUGUAAACUUGACCACAACUACUGUUUUUUUGAAAUACAUGAUUCAUGGUUUACAUGUGUCAAGGUGAAAUCUGAGUUGGCUUUUACAGAUAGUUGACUUUCUAUCUUUUGGCAUUCUUUGGUGUGUAGAAUUACUGUAAUACUUCUGCAAUCAACUGAAAACUAGAGCCUUUAAAUGAUUUCAAUUCCACAGAAAGAAAGUGAGCUUGAACAUAGGAUGAGCUUUAGAAAGAAAAUUGAUCAAGCAGAUGUUUAAUUGGAAUUGAUUAUUAGAUCCUACUUUGUGGAUUUAGUCCCUGGGAUUCAGUCUGUAGAAAUGUCUAAUAGUUCUCUAUAGUCCUUGUUCCUGGUGAACCACAGUUAGGGUGUUUUGUUUAUUUUAUUGUUCUUGCUAUUGUUGAUAUUCUAUGUAGUUGAGCUCUGUAAAAGGAAAUUGUAUUUUAUGUUUUAGUAAUUGUUGCCAACUUUUUAAAUUAAUUUUCAUUAUUUUUGAGCCAAAUUGAAAUGUGCACCUCCUGUGCCUUUUUUCUCCUUAGAAAAUCUAAUUACUUGGAACAAGUUCAGAUUUCACUGGUCAGUCAUUUUCAUCUUGUUUUCUUCUUGCUAAGUCUUACCAUGUACCUGCUUUGGCAAUCAUUGCAACUCUGAGAUUAUAAAAUGCCUUAGAGAAUAUACUAACUAAUAAGAUCUUUUUUUCAGAAACAGAAAAUAGUUCCUUGAGUACUUCCUUCUUGCAUUUCUGCCUAUGUUUUUGAAGUUGUUGCUGUUUGCCUGCAAUAGGCUAUAAGGAAUAGCAGGAGAAAUUUUACUGAAGUGCUGUUUUCCUAGGUGCUACUUUGGCAGAGCUAAGUUAUCUUUUGUUUUCUUAAUGCGUUUGGACCAUUUUGCUGGCUAUAAAAUAACUGAUUAAUAUAAUUCUAACACAAUGUUGACAUUGUAGUUACACAAACACAAAUAAAUAUUUUAUUUAAAAUUCUGGAAGUAAUAUAAAAGGGAAAAUAUAUUUAUAAGAAAGGGAUAAAGGUAAUAGAGCCCUUCUGCCCCCCACCCACCAAAUUUACACAACAAAAUGACAUGUUCGAAUGUGAAAGGUCAUAAUAGCUUUCCCAUCAUGAAUCAGAAAGAUGUGGACAGCUUGAUGUUUUAGACAACCACUGAACUAGAUGACUGUUGUACUGUAGCUCAGUCAUUUAAAAAAUAUAUAAAUACUACCUUGUAGUGUCCCAUACUGUGUUUUUUACAUGGUAGAUUCUUAUUUAAGUGCUAACUGGUUAUUUUCUUUGGCUGGUUUAUUGUACUGUUAUACAGAAUGUAAGUUGUACAGUGAAAUAAGUUAUUAAAGCAUGUGUAAACAUUGUUAUAUAUCUUUUCUCCUAAAUGGAGAAUUUUGAAUAAAAUAUAUUUGAAAUUUUAAAAAAAAAAA AAAAAAA 166 NM_145005.6ACGUAACCUACGGUGUCCCGCUAGGAAAGAGAGGUGCGUCAAACAGCGACAAGUUCCGCCCACGUAAAAGAUGACGCUUGAUAUCUCCGGAGCAUUUGGAUAAUGUGACAGUUGGAAUGCAGUGAUGUCGACUCUUUGCCCACCGCCAUCUCCAGCUGUUGCCAAGACAGAGAUUGCUUUAAGUGGCAAAUCACCUUUAUUAGCAGCUACUUUUGCUUACUGGGACAAUAUUCUUGGUCCUAGAGUAAGGCACAUUUGGGCUCCAAAGACAGAACAGGUACUUCUCAGUGAUGGAGAAAUAACUUUUCUUGCCAACCACACUCUAAAUGGAGAAAUCCUUCGAAAUGCAGAGAGUGGUGCUAUAGAUGUAAAGUUUUUUGUCUUGUCUGAAAAGGGAGUGAUUAUUGUUUCAUUAAUCUUUGAUGGAAACUGGAAUGGGGAUCGCAGCACAUAUGGACUAUCAAUUAUACUUCCACAGACAGAACUUAGUUUCUACCUCCCACUUCAUAGAGUGUGUGUUGAUAGAUUAACACAUAUAAUCCGGAAAGGAAGAAUAUGGAUGCAUAAGGAAAGACAAGAAAAUGUCCAGAAGAUUAUCUUAGAAGGCACAGAGAGAAUGGAAGAUCAGGGUCAGAGUAUUAUUCCAAUGCUUACUGGAGAAGUGAUUCCUGUAAUGGAACUGCUUUCAUCUAUGAAAUCACACAGUGUUCCUGAAGAAAUAGAUAUAGCUGAUACAGUACUCAAUGAUGAUGAUAUUGGUGACAGCUGUCAUGAAGGCUUUCUUCUCAAGUAAGAAUUUUUCUUUUCAUAAAAGCUGGAUGAAGCAGAUACCAUCUUAUGCUCACCUAUGACAAGAUUUGGAAGAAAGAAAAUAACAGACUGUCUACUUAGAUUGUUCUAGGGACAUUACGUAUUUGAACUGUUGCUUAAAUUUGUGUUAUUUUUCACUCAUUAUAUUUCUAUAUAUAUUUGGUGUUAUUCCAUUUGCUAUUUAAAGAAACCGAGUUUCCAUCCCAGACAAGAAAUCAUGGCCCCUUGCUUGAUUCUGGUUUCUUGUUUUACUUCUCAUUAAAGCUAACAGAAUCCUUUCAUAUUAAGUUGUACUGUAGAUGAACUUAAGUUAUUUAGGCGUAGAACAAAAUUAUUCAUAUUUAUACUGAUCUUUUUCCAUCCAGCAGUGGAGUUUAGUACUUAAGAGUUUGUGCCCUUAAACCAGACUCCCUGGAUUAAUGCUGUGUACCCGUGGGCAAGGUGCCUGAAUUCUCUAUACACCUAUUUCCUCAUCUGUAAAAUGGCAAUAAUAGUAAUAGUACCUAAUGUGUAGGGUUGUUAUAAGCAUUGAGUAAGAUAAAUAAUAUAAAGCACUUAGAACAGUGCCUGGAACAUAAAAACACUUAAUAAUAGCUCAUAGCUAACAUUUCCUAUUUACAUUUCUUCUAGAAAUAGCCAGUAUUUGUUGAGUGCCUACAUGUUAGUUCCUUUACUAGUUGCUUUACAUGUAUUAUCUUAUAUUCUGUUUUAAAGUUUCUUCACAGUUACAGAUUUUCAUGAAAUUUUACUUUUAAUAAAAGAGAAGUAAAAGUAUAAAGUAUUCACUUUUAUGUUCACAGUCUUUUCCUUUAGGCUCAUGAUGGAGUAUCAGAGGCAUGAGUGUGUUUAACCUAAGAGCCUUAAUGGCUUGAAUCAGAAGCACUUUAGUCCUGUAUCUGUUCAGUGUCAGCCUUUCAUACAUCAUUUUAAAUCCCAUUUGACUUUAAGUAAGUCACUUAAUCUCUCUACAUGUCAAUUUCUUCAGCUAUAAAAUGAUGGUAUUUCAAUAAAUAAAUACAUUAAUUAAAUGAUAUUAUACUGACUAAUUGGGCUGUUUUAAGGCUCAAUAAGAAAAUUUCUGUGAAAGGUCUCUAGAAAAUGUAGGUUCCUAUACAAAUAAAAGAUAACAUUGUGCUU AUAAAAAAAA

Interfering RNA

Using the compositions and methods described herein, a patient having aspliceopathy and/or a disorder characterized by RNA dominance may beadministered an interfering RNA molecule, a composition containing thesame, or a vector encoding the same, so as to suppress the expression ofa mutant RNA transcript containing an expanded repeat region. Forexample, in the case of myotonic dystrophy type I, the target RNAtranscript to be suppressed is human DMPK RNA containing expanded CUGrepeat regions in the 3′ UTR of the transcript. In the case of myotonicdystrophy type II, the target RNA transcript to be suppressed is humanZNF9 RNA containing expanded CCUG (SEQ ID NO: 164) repeat regions. Inthe case of amyotrophic lateral sclerosis, the target RNA transcript tobe suppressed is human C9ORF72 RNA containing expanded GGGGCC (SEQ IDNO: 162) repeat regions.

Exemplary interfering RNA molecules that may be used in conjunction withthe compositions and methods described herein for the treatment of RNAdominance disorders, such as myotonic dystrophy type I and others, aresiRNA molecules, miRNA molecules, and shRNA molecules, among others. Inthe case of siRNA molecules, the siRNA may be single stranded or doublestranded. miRNA molecules, in contrast, are single-stranded moleculesthat form a hairpin, thereby adopting a hydrogen-bonded structurereminiscent of a nucleic acid duplex. In either case, the interferingRNA may contain an antisense or “guide” strand that anneals (e.g., byway of complementarity) to the repeat-expanded mutant RNA target. Theinterfering RNA may also contain a “passenger” strand that iscomplementary to the guide strand and, thus, may have the same nucleicacid sequence as the RNA target.

Exemplary interfering RNA molecules that anneal to mutant DMPKcontaining expanded CUG repeat motifs and that may be used inconjunction with the compositions and methods described herein for thetreatment of myotonic dystrophy type I are shown in Table 4, below. Agraphical representation of the sites on a target DMPK RNA transcript towhich the following interfering RNA molecules anneal by way of sequencecomplementarity is shown in FIG. 1.

TABLE 4 Exemplary RNAi molecules useful for suppressing mutant DMPKRNA expression SEQ ID Type of Interfering NO. RNA Nucleic Acid Sequence3 siRNA (Identical to CGCCAGUCAACUACGCGA target sequence) 4siRNA (Identical to CCUGCUCCUGUUCGCCGUU target sequence) 5siRNA (Identical to CCCGACAUUCCUCGGUAUU target sequence) 6siRNA (Identical to CCUAGAACUGUCUUCGACU target sequence) 7siRNA (Identical to CCUAUCGUUGGUUCGCAAA target sequence) 8siRNA (Identical to GCUGGGUGUAUUCGCCUAU target sequence) 9siRNA (Identical to GGACAUCAAACCCGACAAC target sequence) 10siRNA (Identical to GCGGGCAGAUGGAACGGUG target sequence) 11siRNA (Identical to GGCCUAUCGGAGGCGCUUU target sequence) 12siRNA (Identical to CCCUAGAACUGUCUUCGAC target sequence) 13siRNA (Identical to GGACAACCAGAACUUCGCC target sequence) 14siRNA (Identical to CGCUGGGUGUAUUCGCCUA target sequence) 15siRNA (Identical to CACCUAUCGUUGGUUCGCA target sequence) 16siRNA (Identical to GGCGCUGGGUGUAUUCGCC target sequence) 17siRNA (Identical to CGCCCUUCUACGCGGAUUC target sequence) 18siRNA (Identical to CGAAGGUGCCACCGACACA target sequence) 19siRNA (Identical to CCAUUUCUUUCUUUCGGCC target sequence) 20siRNA (Identical to GCCGUUGUUCUGUCUCGUG target sequence) 21siRNA (Identical to GCUCCUGUUCGCCGUUGUU target sequence) 22siRNA (Identical to GCCAGUUCACAACCGCUCC target sequence) 23siRNA (Identical to CCAGUUCACAACCGCUCCG target sequence) 24siRNA (Identical to GGUCCUUGUAGCCGGGAAU target sequence) 25siRNA (Identical to CCACAGUCAACUACGCGAG target sequence) 26siRNA (Identical to CGACCUCAGGUAGCGGUAG target sequence) 27siRNA (Identical to CGGGAAGGUGCGCCGCUAG target sequence) 28siRNA (Identical to GGGAUUCGAGGCGUGCGAG target sequence) 29siRNA (Identical to GGCUUAAGGAGGUCCGACU target sequence) 30siRNA (Identical to CCCUACGUCUUUGCGACUU target sequence) 31siRNA (Identical to CGACUGCAGAGGGACGACU target sequence) 32siRNA (Identical to CCAAUGACGAGUUCGGACG target sequence) 33siRNA (Identical to GGUCCAGGUGCGGUCGCUG target sequence) 34siRNA (Identical to GCAGGAGACACUGUCGGAC target sequence) 35siRNA (Identical to CCCACGUCUGGGCCGUUAC target sequence) 36siRNA (Identical to GCACCGGCUUGGCUACGUG target sequence) 37siRNA (Identical to GCUUGAUUCUGAACCGCUG target sequence) 38siRNA (Identical to GCUUAAGGAGGUCCGACUG target sequence) 39siRNA (Identical to CGUGCUGUCACUGGACGAG target sequence) 40miRNA (passenger AGCGCCAGUCAACUACGCGAG strand) 41 miRNA (passengerCCCCUGCUCCUGUUCGCCGUU strand) 42 miRNA (passenger AGCCCGACAUUCCUCGGUAUUstrand) 43 miRNA (passenger ACCCUAGAACUGUCUUCGAUU strand) 44miRNA (passenger ACCCUAUCGUUGGUUCGCAAA strand) 45 miRNA (passengerACGCUGGGUGUAUUCGCCUAU strand) 46 miRNA (passenger CGGGACAUCAAACCCGACAAUstrand) 47 miRNA (passenger ACGCGGGCAGAUGGAACGGUG strand) 48miRNA (passenger ACGGCCUAUCGGAGGCGCUUU strand) 49 miRNA (passengerAGCCCUAGAACUGUCUUCGAU strand) 50 miRNA (passenger ACGGACAACCAGAACUUCGUUstrand) 51 miRNA (passenger AGCGCUGGGUGUAUUCGCUUA strand) 52miRNA (passenger ACCACCUAUCGUUGGUUCGUA strand) 53 miRNA (passengerCGGGCGCUGGGUGUAUUCGUU strand) 54 miRNA (passenger ACCGCCCUUCUACGCGGAUUUstrand) 55 miRNA (passenger CGCGAAGGUGCCACCGACAUA strand) 56miRNA (passenger ACCCAUUUCUUUCUUUCGGUU strand) 57 miRNA (passengerACGCCGUUGUUCUGUCUCGUG strand) 58 miRNA (passenger ACGCUCCUGUUCGCCGUUGUUstrand) 59 miRNA (passenger ACGCCAGUUCACAACCGCUUU strand) 60miRNA (passenger CGCCAGUUCACAACCGCUUUG strand) 61 miRNA (passengerCGGGUCCUUGUAGCCGGGAAU strand) 62 miRNA (passenger ACCCACAGUCAACUACGCGAGstrand) 63 miRNA (passenger ACCGACCUCAGGUAGCGGUAG strand) 64miRNA (passenger CGCGGGAAGGUGCGCCGCUAG strand) 65 miRNA (passengerACGGGAUUCGAGGCGUGCGAG strand) 66 miRNA (passenger ACGGCUUAAGGAGGUCCGAUUstrand) 67 miRNA (passenger AGCCCUACGUCUUUGCGAUUU strand) 68miRNA (passenger CCCGACUGCAGAGGGACGAUU strand) 69 miRNA (passengerCGCCAAUGACGAGUUCGGAUG strand) 70 miRNA (passenger CGGGUCCAGGUGCGGUCGUUGstrand) 71 miRNA (passenger ACGCAGGAGACACUGUCGGAU strand) 72miRNA (passenger CGCCCACGUCUGGGCCGUUAU strand) 73 miRNA (passengerACGCACCGGCUUGGCUACGUG strand) 74 miRNA (passenger ACGCUUGAUUCUGAACCGUUGstrand) 75 miRNA (passenger CGGCUUAAGGAGGUCCGAUUG strand) 76miRNA (passenger ACCGUGCUGUCACUGGACGAG strand) 77 miRNA (guide strand)UUCGCGUAGUUGACUGGCGCC 78 miRNA (guide strand) AACGGCGAACAGGAGCAGGGC 79miRNA (guide strand) AAUACCGAGGAAUGUCGGGCC 80 miRNA (guide strand)AGUCGAAGACAGUUCUAGGGC 81 miRNA (guide strand) UUUGCGAACCAACGAUAGGGC 82miRNA (guide strand) AUAGGCGAAUACACCCAGCGC 83 miRNA (guide strand)GUUGUCGGGUUUGAUGUCCCA 84 miRNA (guide strand) UACCGUUCCAUCUGCCCGCGC 85miRNA (guide strand) AAAGCGCCUCCGAUAGGCCGC 86 miRNA (guide strand)GUCGAAGACAGUUCUAGGGCC 87 miRNA (guide strand) GGCGAAGUUCUGGUUGUCCGC 88miRNA (guide strand) UAGGCGAAUACACCCAGCGCC 89 miRNA (guide strand)UGCGAACCAACGAUAGGUGGC 90 miRNA (guide strand) GGCGAAUACACCCAGCGCCCA 91miRNA (guide strand) GAAUCCGCGUAGAAGGGCGGC 92 miRNA (guide strand)UGUGUCGGUGGCACCUUCGCA 93 miRNA (guide strand) GGCCGAAAGAAAGAAAUGGGC 94miRNA (guide strand) UACGAGACAGAACAACGGCGC 95 miRNA (guide strand)AACAACGGCGAACAGGAGCGC 96 miRNA (guide strand) GGAGCGGUUGUGAACUGGCGC 97miRNA (guide strand) UGGAGCGGUUGUGAACUGGCA 98 miRNA (guide strand)AUUCCCGGCUACAAGGACCCU 99 miRNA (guide strand) UUCGCGUAGUUGACUGUGG 100miRNA (guide strand) UUACCGCUACCUGAGGUCGGC 101 miRNA (guide strand)UUAGCGGCGCACCUUCCCGCA 102 miRNA (guide strand) UUCGCACGCCUCGAAUCCCGC 103miRNA (guide strand) AGUCGGACCUCCUUAAGCCGC 104 miRNA (guide strand)AAGUCGCAAAGACGUAGGGCC 105 miRNA (guide strand) AGUCGUCCCUCUGCAGUCGGA 106miRNA (guide strand) UGUCCGAACUCGUCAUUGGCA 107 miRNA (guide strand)UAGCGACCGCACCUGGACCCA 108 miRNA (guide strand) GUCCGACAGUGUCUCCUGCGC 109miRNA (guide strand) GUAACGGCCCAGACGUGGGCA 110 miRNA (guide strand)UACGUAGCCAAGCCGGUGCGC 111 miRNA (guide strand) UAGCGGUUCAGAAUCAAGCGC 112miRNA (guide strand) UAGUCGGACCUCCUUAAGCCA 113 miRNA (guide strand)UUCGUCCAGUGACAGCACGGC 114 miRNA AAAACUCGAGUGAGCGCGGGCGCUGGGUGUAUUCGUUACUGUAAAGCCACAGAUG 115 miRNA AAAACUCGAGUGAGCGACCGCCCUUCUACGCGGAUUUACUGUAAAGCCACAGAUG 116 miRNA AAAACUCGAGUGAGCGCGCGAAGGUGCCACCGACAUAACUGUAAAGCCACAGAUG 117 miRNA AAAACUCGAGUGAGCGACCCAUUUCUUUCUUUCGGUUACUGUAAAGCCACAGAUG 118 miRNA AAAACUCGAGUGAGCGACGCCGUUGUUCUGUCUCGUGACUGUAAAGCCACAGAUG 119 miRNA AAAACUCGAGUGAGCGACGCUCCUGUUCGCCGUUGUUACUGUAAAGCCACAGAUG 120 miRNA AAAACUCGAGUGAGCGACGCCAGUUCACAACCGCUUUACUGUAAAGCCACAGAUG 121 miRNA AAAACUCGAGUGAGCGCGCCAGUUCACAACCGCUUUGACUGUAAAGCCACAGAUG 122 miRNA AAAACUCGAGUGAGCGCGGGUCCUUGUAGCCGGGAAUACUGUAAAGCCACAGAUG 123 miRNA UUUUACUAGUAGGCGUGGGCGCUGGGUGUAUUCGCCAGCAUCUGUGGCUUUACAG 124 miRNA UUUUACUAGUAGGCGGCCGCCCUUCUACGCGGAUUCAGCAUCUGUGGCUUUACAG 125 miRNA UUUUACUAGUAGGCGUGCGAAGGUGCCACCGACACAAGCAUCUGUGGCUUUACAG 126 miRNA UUUUACUAGUAGGCGGCCCAUUUCUUUCUUUCGGCCAGCAUCUGUGGCUUUACAG 127 miRNA UUUUACUAGUAGGCGGCGCCGUUGUUCUGUCUCGUAAGCAUCUGUGGCUUUACAG 128 miRNA UUUUACUAGUAGGCGGCGCUCCUGUUCGCCGUUGUUAGCAUCUGUGGCUUUACAG 129 miRNA UUUUACUAGUAGGCGGCGCCAGUUCACAACCGCUCCAGCAUCUGUGGCUUUACAG 130 miRNA UUUUACUAGUAGGCGUGCCAGUUCACAACCGCUCCAAGCAUCUGUGGCUUUACAG 131 miRNA UUUUACUAGUAGGCGUGGGUCCUUGUAGCCGGGAAUAGCAUCUGUGGCUUUACAG 132 miRNA AAAACUCGAGUGAGCGACCCACAGUCAACUACGCGAGACUGUAAAGCCACAGAUG 133 miRNA UUUUACUAGUAGGCGGCCCACAGUCAACUACGCGAAAGCAUCUGUGGCUUUACAG 134 miRNA AAAACUCGAGUGAGCGACCGACCUCAGGUAGCGGUAGACUGUAAAGCCACAGAUG 135 miRNA UUUUACUAGUAGGCGGCCGACCUCAGGUAGCGGUAAAGCAUCUGUGGCUUUACAG 136 miRNA AAAACUCGAGUGAGCGCGCGGGAAGGUGCGCCGCUAGACUGUAAAGCCACAGAUG 137 miRNA UUUUACUAGUAGGCGUGCGGGAAGGUGCGCCGCUAAAGCAUCUGUGGCUUUACAG 138 miRNA AAAACUCGAGUGAGCGACGGGAUUCGAGGCGUGCGAGACUGUAAAGCCACAGAUG 139 miRNA UUUUACUAGUAGGCGGCGGGAUUCGAGGCGUGCGAAAGCAUCUGUGGCUUUACAG 140 miRNA AAAACUCGAGUGAGCGACGGCUUAAGGAGGUCCGAUUACUGUAAAGCCACAGAUG 141 miRNA UUUUACUAGUAGGCGGCGGCUUAAGGAGGUCCGACUAGCAUCUGUGGCUUUACAG 142 miRNA AAAACUCGAGUGAGCGAGCCCUACGUCUUUGCGAUUUACUGUAAAGCCACAGAUG 143 miRNA UUUUACUAGUAGGCGGGCCCUACGUCUUUGCGACUUAGCAUCUGUGGCUUUACAG 144 miRNA AAAACUCGAGUGAGCGCCCGACUGCAGAGGGACGAUUACUGUAAAGCCACAGAUG 145 miRNA UUUUACUAGUAGGCGUCCGACUGCAGAGGGACGACUAGCAUCUGUGGCUUUACAG 146 miRNA AAAACUCGAGUGAGCGCGCCAAUGACGAGUUCGGAUGACUGUAAAGCCACAGAUG 147 miRNA UUUUACUAGUAGGCGUGCCAAUGACGAGUUCGGACAAGCAUCUGUGGCUUUACAG 148 miRNA AAAACUCGAGUGAGCGCGGGUCCAGGUGCGGUCGUUGACUGUAAAGCCACAGAUG 149 miRNA UUUUACUAGUAGGCGUGGGUCCAGGUGCGGUCGCUAAGCAUCUGUGGCUUUACAG 150 miRNA AAAACUCGAGUGAGCGACGCAGGAGACACUGUCGGAUACUGUAAAGCCACAGAUG 151 miRNA UUUUACUAGUAGGCGGCGCAGGAGACACUGUCGGACAGCAUCUGUGGCUUUACAG 152 miRNA AAAACUCGAGUGAGCGCGCCCACGUCUGGGCCGUUAUACUGUAAAGCCACAGAUG 153 miRNA UUUUACUAGUAGGCGUGCCCACGUCUGGGCCGUUACAGCAUCUGUGGCUUUACAG 154 miRNA AAAACUCGAGUGAGCGACGCACCGGCUUGGCUACGUGACUGUAAAGCCACAGAUG 155 miRNA UUUUACUAGUAGGCGGCGCACCGGCUUGGCUACGUAAGCAUCUGUGGCUUUACAG 156 miRNA AAAACUCGAGUGAGCGACGCUUGAUUCUGAACCGUUGACUGUAAAGCCACAGAUG 157 miRNA UUUUACUAGUAGGCGGCGCUUGAUUCUGAACCGCUAAGCAUCUGUGGCUUUACAG 158 miRNA AAAACUCGAGUGAGCGCGGCUUAAGGAGGUCCGAUUGACUGUAAAGCCACAGAUG 159 miRNA UUUUACUAGUAGGCGUGGCUUAAGGAGGUCCGACUAAGCAUCUGUGGCUUUACAG 160 miRNA AAAACUCGAGUGAGCGACCGUGCUGUCACUGGACGAGACUGUAAAGCCACAGAUG 161 miRNA UUUUACUAGUAGGCGGCCGUGCUGUCACUGGACGAAAGCAUCUGUGGCUUUACAGExemplary miRNA constructs useful in conjunction with the compositionsand methods described herein are those that have a combination ofpassenger and guide strands shown in Table 5, below.

TABLE 5 Exemplary anti-DMPK miRNA guide strand/passenger strandcombinations Entry siRNA on which miRNA miRNA miRNA No. construct isbased Passenger Strand Guide Strand 1 SEQ ID NO: 3 SEQ ID NO: 40 SEQ IDNO: 77 2 SEQ ID NO: 4 SEQ ID NO: 41 SEQ ID NO: 78 3 SEQ ID NO: 5 SEQ IDNO: 42 SEQ ID NO: 79 4 SEQ ID NO: 6 SEQ ID NO: 43 SEQ ID NO: 80 5 SEQ IDNO: 7 SEQ ID NO: 44 SEQ ID NO: 81 6 SEQ ID NO: 8 SEQ ID NO: 45 SEQ IDNO: 82 7 SEQ ID NO: 9 SEQ ID NO: 46 SEQ ID NO: 83 8 SEQ ID NO: 10 SEQ IDNO: 47 SEQ ID NO: 84 9 SEQ ID NO: 11 SEQ ID NO: 48 SEQ ID NO: 85 10 SEQID NO: 12 SEQ ID NO: 49 SEQ ID NO: 86 11 SEQ ID NO: 13 SEQ ID NO: 50 SEQID NO: 87 12 SEQ ID NO: 14 SEQ ID NO: 51 SEQ ID NO: 88 13 SEQ ID NO: 15SEQ ID NO: 52 SEQ ID NO: 89 14 SEQ ID NO: 16 SEQ ID NO: 53 SEQ ID NO: 9015 SEQ ID NO: 17 SEQ ID NO: 54 SEQ ID NO: 91 16 SEQ ID NO: 18 SEQ ID NO:55 SEQ ID NO: 92 17 SEQ ID NO: 19 SEQ ID NO: 56 SEQ ID NO: 93 18 SEQ IDNO: 20 SEQ ID NO: 57 SEQ ID NO: 94 19 SEQ ID NO: 21 SEQ ID NO: 58 SEQ IDNO: 95 20 SEQ ID NO: 22 SEQ ID NO: 59 SEQ ID NO: 96 21 SEQ ID NO: 23 SEQID NO: 60 SEQ ID NO: 97 22 SEQ ID NO: 24 SEQ ID NO: 61 SEQ ID NO: 98 23SEQ ID NO: 25 SEQ ID NO: 62 SEQ ID NO: 99 24 SEQ ID NO: 26 SEQ ID NO: 63SEQ ID NO: 100 25 SEQ ID NO: 27 SEQ ID NO: 64 SEQ ID NO: 101 26 SEQ IDNO: 28 SEQ ID NO: 65 SEQ ID NO: 102 27 SEQ ID NO: 29 SEQ ID NO: 66 SEQID NO: 103 28 SEQ ID NO: 30 SEQ ID NO: 67 SEQ ID NO: 104 29 SEQ ID NO:31 SEQ ID NO: 68 SEQ ID NO: 105 30 SEQ ID NO: 32 SEQ ID NO: 69 SEQ IDNO: 106 31 SEQ ID NO: 33 SEQ ID NO: 70 SEQ ID NO: 107 32 SEQ ID NO: 34SEQ ID NO: 71 SEQ ID NO: 108 33 SEQ ID NO: 35 SEQ ID NO: 72 SEQ ID NO:109 34 SEQ ID NO: 36 SEQ ID NO: 73 SEQ ID NO: 110 35 SEQ ID NO: 37 SEQID NO: 74 SEQ ID NO: 111 36 SEQ ID NO: 38 SEQ ID NO: 75 SEQ ID NO: 11237 SEQ ID NO: 39 SEQ ID NO: 76 SEQ ID NO: 113

Vectors for Delivery of Interfering RNA Viral Vectors for InterferingRNA Delivery

Viral genomes provide a rich source of vectors that can be used for theefficient delivery of a gene of interest into the genome of a targetcell in a patient (e.g., a mammalian cell, such as a human cell). Viralgenomes are particularly useful vectors for gene delivery because thepolynucleotides contained within such genomes are typically incorporatedinto the genome of a target cell by generalized or specializedtransduction. These processes occur as part of the natural viralreplication cycle, and do not require added proteins or reagents inorder to induce gene integration. Examples of viral vectors that may beused in conjunction with the compositions and methods described hereinare AAV, retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48),parvovirus (e.g., adeno-associated viruses), coronavirus, negativestrand RNA viruses such as orthomyxovirus (e.g., influenza virus),rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus(e.g. measles and Sendai), positive strand RNA viruses, such aspicornavirus and alphavirus, and double stranded DNA viruses includingadenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2,Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia,modified vaccinia Ankara (MVA), fowlpox and canarypox). Other virusesthat may be used in conjunction with the compositions and methodsdescribed herein include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosis-sarcoma, mammalianC-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Otherexamples include murine leukemia viruses, murine sarcoma viruses, mousemammary tumor virus, bovine leukemia virus, feline leukemia virus,feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus,baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkeyvirus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcomavirus and lentiviruses. Other examples of vectors are described, forexample, in U.S. Pat. No. 5,801,030, the disclosure of which isincorporated herein by reference as it pertains to viral vectors for usein gene therapy.

AAV Vectors for Interfering RNA Delivery

In some embodiments, interfering RNA constructs described herein areincorporated into recombinant AAV (rAAV) vectors in order to facilitatetheir introduction into a cell, such as a target cardiac cell (e.g., amuscle cell) in a patient. rAAV vectors useful in the conjunction withthe compositions and methods described herein include recombinantnucleic acid constructs that contain (1) a transgene encoding aninterfering RNA construct described herein (such as an siRNA, shRNA, ormiRNA described herein) and (2) nucleic acids that facilitate andexpression of the heterologous genes. The viral nucleic acids mayinclude those sequences of AAV that are required in cis for replicationand packaging (e.g., functional ITRs) of the DNA into a virion. SuchrAAV vectors may also contain marker or reporter genes. Useful rAAVvectors include those having one or more of the naturally-occurring AAVgenes deleted in whole or in part, but retain functional flanking ITRsequences. The AAV ITRs may be of any serotype (e.g., derived fromserotype 2) suitable for a particular application. Methods for usingrAAV vectors are described, for example, in Tal et al., J. Biomed. Sci.7:279-291 (2000), and Monahan and Samulski, Gene Delivery 7:24-30(2000), the disclosures of each of which are incorporated herein byreference as they pertain to AAV vectors for gene delivery.

The nucleic acids and vectors described herein can be incorporated intoa rAAV virion in order to facilitate introduction of the nucleic acid orvector into a cell. The capsid proteins of AAV compose the exterior,non-nucleic acid portion of the virion and are encoded by the AAV capgene. The cap gene encodes three viral coat proteins, VP1, VP2 and VP3,which are required for virion assembly. The construction of rAAV virionshas been described, for example, in U.S. Pat. Nos. 5,173,414; 5,139,941;5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as in Rabinowitzet al., J. Virol. 76:791-801 (2002) and Bowles et al., J. Virol.77:423-432 (2003), the disclosures of each of which are incorporatedherein by reference as they pertain to AAV vectors for gene delivery.

rAAV virions useful in conjunction with the compositions and methodsdescribed herein include those derived from a variety of AAV serotypesincluding AAV 1, 2, 3, 4, 5, 6, 7, 8 and 9. Construction and use of AAVvectors and AAV proteins of different serotypes are described, forexample, in Chao et al., Mol. Ther. 2:619-623 (2000); Davidson et al.,Proc. Natl. Acad. Sci. USA 97:3428-3432 (2000); Xiao et al., J. Virol.72:2224-2232 (1998); Halbert et al., J. Virol. 74:1524-1532 (2000);Halbert et al., J. Virol. 75:6615-6624 (2001); and Auricchio et al.,Hum. Molec. Genet. 10:3075-3081 (2001), the disclosures of each of whichare incorporated herein by reference as they pertain to AAV vectors forgene delivery.

Also useful in conjunction with the compositions and methods describedherein are pseudotyped rAAV vectors. Pseudotyped vectors include AAVvectors of a given serotype (e.g., AAV2) pseudotyped with a capsid genederived from a serotype other than the given serotype (e.g., AAV1, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, among others). For example, arepresentative pseudotyped vector is an AAV2 vector encoding atherapeutic protein pseudotyped with a capsid gene derived from AAVserotype 8 or AAV serotype 9. Techniques involving the construction anduse of pseudotyped rAAV virions are known in the art and are described,for example, in Duan et al., J. Virol. 75:7662-7671 (2001); Halbert etal., J. Virol. 74:1524-1532 (2000); Zolotukhin et al., Methods,28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet.,10:3075-3081 (2001).

AAV virions that have mutations within the virion capsid may be used toinfect particular cell types more effectively than non-mutated capsidvirions. For example, suitable AAV mutants may have ligand insertionmutations for the facilitation of targeting AAV to specific cell types.The construction and characterization of AAV capsid mutants includinginsertion mutants, alanine screening mutants, and epitope tag mutants isdescribed in Wu et al., J. Virol. 74:8635-45 (2000). Other rAAV virionsthat can be used in methods of the invention include those capsidhybrids that are generated by molecular breeding of viruses as well asby exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436-439(2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423-428 (2001).

Additional Methods for the Delivery of Interfering RNA TransfectionTechniques

Techniques that can be used to introduce a transgene, such as atransgene encoding an interfering RNA construct described herein, into atarget cell (e.g., a target cell from or within a human patientsuffering from RNA dominance) are known in the art. For example,electroporation can be used to permeabilize mammalian cells (e.g., humantarget cells) by the application of an electrostatic potential to thecell of interest. Mammalian cells, such as human cells, subjected to anexternal electric field in this manner are subsequently predisposed tothe uptake of exogenous nucleic acids. Electroporation of mammaliancells is described in detail, e.g., in Chu et al., Nucleic AcidsResearch 15:1311 (1987), the disclosure of which is incorporated hereinby reference. A similar technique, Nucleofection™, utilizes an appliedelectric field in order to stimulate the uptake of exogenouspolynucleotides into the nucleus of a eukaryotic cell. Nucleofection™and protocols useful for performing this technique are described indetail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005),as well as in US 2010/0317114, the disclosures of each of which areincorporated herein by reference.

Additional techniques useful for the transfection of target cellsinclude the squeeze-poration methodology. This technique induces therapid mechanical deformation of cells in order to stimulate the uptakeof exogenous DNA through membranous pores that form in response to theapplied stress. This technology is advantageous in that a vector is notrequired for delivery of nucleic acids into a cell, such as a humantarget cell. Squeeze-poration is described in detail, e.g., in Sharei etal., Journal of Visualized Experiments 81:e50980 (2013), the disclosureof which is incorporated herein by reference.

Lipofection represents another technique useful for transfection oftarget cells. This method involves the loading of nucleic acids into aliposome, which often presents cationic functional groups, such asquaternary or protonated amines, towards the liposome exterior. Thispromotes electrostatic interactions between the liposome and a cell dueto the anionic nature of the cell membrane, which ultimately leads touptake of the exogenous nucleic acids, for example, by direct fusion ofthe liposome with the cell membrane or by endocytosis of the complex.Lipofection is described in detail, for example, in U.S. Pat. No.7,442,386, the disclosure of which is incorporated herein by reference.Similar techniques that exploit ionic interactions with the cellmembrane to provoke the uptake of foreign nucleic acids includecontacting a cell with a cationic polymer-nucleic acid complex.Exemplary cationic molecules that associate with polynucleotides so asto impart a positive charge favorable for interaction with the cellmembrane are activated dendrimers (described, e.g., in Dennig, Topics inCurrent Chemistry 228:227 (2003), the disclosure of which isincorporated herein by reference) and diethylaminoethyl (DEAE)-dextran,the use of which as a transfection agent is described in detail, forexample, in Gulick et al., Current Protocols in Molecular Biology40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein byreference. Magnetic beads are another tool that can be used to transfecttarget cells in a mild and efficient manner, as this methodologyutilizes an applied magnetic field in order to direct the uptake ofnucleic acids. This technology is described in detail, for example, inUS 2010/0227406, the disclosure of which is incorporated herein byreference.

Another useful tool for inducing the uptake of exogenous nucleic acidsby target cells is laserfection, a technique that involves exposing acell to electromagnetic radiation of a particular wavelength in order togently permeabilize the cells and allow polynucleotides to penetrate thecell membrane. This technique is described in detail, e.g., in Rhodes etal., Methods in Cell Biology 82:309 (2007), the disclosure of which isincorporated herein by reference.

Microvesicles represent another potential vehicle that can be used tomodify the genome of a target cell according to the methods describedherein. For example, microvesicles that have been induced by theco-overexpression of the glycoprotein VSV-G with, e.g., agenome-modifying protein, such as a nuclease, can be used to efficientlydeliver proteins into a cell that subsequently catalyze thesite-specific cleavage of an endogenous polynucleotide sequence so as toprepare the genome of the cell for the covalent incorporation of apolynucleotide of interest, such as a gene or regulatory sequence. Theuse of such vesicles, also referred to as Gesicles, for the geneticmodification of eukaryotic cells is described in detail, e.g., in Quinnet al., Genetic Modification of Target Cells by Direct Delivery ofActive Protein [abstract]. In: Methylation changes in early embryonicgenes in cancer [abstract], in: Proceedings of the 18th Annual Meetingof the American Society of Gene and Cell Therapy; 2015 May 13, AbstractNo. 122.

Incorporation of Genes Encoding Interfering RNA by Gene Editing

In addition to the above, a variety of tools have been developed thatcan be used for the incorporation of a transgene, such as a transgeneencoding an interfering RNA construct described herein, into a targetcell, and particularly into a human cell. One such method that can beused for incorporating polynucleotides encoding interfering RNAconstructs into target cells involves the use of transposons.Transposons are polynucleotides that encode transposase enzymes andcontain a polynucleotide sequence or gene of interest flanked by 5′ and3′ excision sites. Once a transposon has been delivered into a cell,expression of the transposase gene commences and results in activeenzymes that cleave the gene of interest from the transposon. Thisactivity is mediated by the site-specific recognition of transposonexcision sites by the transposase. In some instances, these excisionsites may be terminal repeats or inverted terminal repeats. Once excisedfrom the transposon, the transgene of interest can be integrated intothe genome of a mammalian cell by transposase-catalyzed cleavage ofsimilar excision sites that exist within the nuclear genome of the cell.This allows the transgene of interest to be inserted into the cleavednuclear DNA at the complementary excision sites, and subsequent covalentligation of the phosphodiester bonds that join the gene of interest tothe DNA of the mammalian cell genome completes the incorporationprocess. In certain cases, the transposon may be a retrotransposon, suchthat the gene encoding the target gene is first transcribed to an RNAproduct and then reverse-transcribed to DNA before incorporation in themammalian cell genome. Exemplary transposon systems are the piggybactransposon (described in detail in, e.g., WO 2010/085699) and thesleeping beauty transposon (described in detail in, e.g., US2005/0112764), the disclosures of each of which are incorporated hereinby reference as they pertain to transposons for use in gene delivery toa cell of interest.

Another tool for the integration of target transgenes into the genome ofa target cell is the clustered regularly interspaced short palindromicrepeats (CRISPR)/Cas system, a system that originally evolved as anadaptive defense mechanism in bacteria and archaea against viralinfection. The CRISPR/Cas system includes palindromic repeat sequenceswithin plasmid DNA and an associated Cas9 nuclease. This ensemble of DNAand protein directs site specific DNA cleavage of a target sequence byfirst incorporating foreign DNA into CRISPR loci. Polynucleotidescontaining these foreign sequences and the repeat-spacer elements of theCRISPR locus are in turn transcribed in a host cell to create a guideRNA, which can subsequently anneal to a target sequence and localize theCas9 nuclease to this site. In this manner, highly site-specificcas9-mediated DNA cleavage can be engendered in a foreign polynucleotidebecause the interaction that brings cas9 within close proximity of thetarget DNA molecule is governed by RNA:DNA hybridization. As a result,one can design a CRISPR/Cas system to cleave any target DNA molecule ofinterest. This technique has been exploited in order to edit eukaryoticgenomes (Hwang et al., Nature Biotechnology 31:227 (2013)) and can beused as an efficient means of site-specifically editing target cellgenomes in order to cleave DNA prior to the incorporation of a geneencoding a target gene. The use of CRISPR/Cas to modulate geneexpression has been described in, for example, U.S. Pat. No. 8,697,359,the disclosure of which is incorporated herein by reference as itpertains to the use of the CRISPR/Cas system for genome editing.Alternative methods for site-specifically cleaving genomic DNA prior tothe incorporation of a transgene of interest in a target cell includethe use of zinc finger nucleases (ZFNs) and transcription activator-likeeffector nucleases (TALENs). Unlike the CRISPR/Cas system, these enzymesdo not contain a guiding polynucleotide to localize to a specific targetsequence. Target specificity is instead controlled by DNA bindingdomains within these enzymes. The use of ZFNs and TALENs in genomeediting applications is described, e.g., in Urnov et al., Nature ReviewsGenetics 11:636 (2010); and in Joung et al., Nature Reviews MolecularCell Biology 14:49 (2013), the disclosure of each of which areincorporated herein by reference as they pertain to compositions andmethods for genome editing.

Additional genome editing techniques that can be used to incorporatepolynucleotides encoding target transgenes into the genome of a targetcell include the use of ARCUS™ meganucleases that can be rationallydesigned so as to site-specifically cleave genomic DNA. The use of theseenzymes for the incorporation of genes encoding target genes into thegenome of a mammalian cell is advantageous in view of the definedstructure-activity relationships that have been established for suchenzymes. Single chain meganucleases can be modified at certain aminoacid positions in order to create nucleases that selectively cleave DNAat desired locations, enabling the site-specific incorporation of atarget transgene into the nuclear DNA of a target cell. Thesesingle-chain nucleases have been described extensively in, for example,U.S. Pat. Nos. 8,021,867 and 8,445,251, the disclosures of each of whichare incorporated herein by reference as they pertain to compositions andmethods for genome editing.

Methods of Detecting RNA Transcript Expression

The expression level of a pathological RNA transcript, such as a DMPKRNA transcript harboring an expanded CUG trinucleotide repeat or aC9ORF72 RNA transcript harboring an expanded GGGGCC (SEQ ID NO: 162)hexanucleotide can be ascertained, for example, by a variety of nucleicacid detection techniques. Additionally or alternatively, RNA transcriptexpression can be inferred by evaluating the concentration or relativeabundance of an encoded protein produced by translation of the RNAtranscript. Protein concentrations can also be assessed, for example,using functional assays. Using these techniques, a reduction in theconcentration of pathological RNA transcripts in response to thecompositions and methods described herein can be observed, whilemonitoring the expression of the encoded protein. The sections thatfollow describe exemplary techniques that can be used to measure theexpression level of a pathological RNA transcript and its downstreamprotein product. RNA transcript expression can be evaluated by a numberof methodologies known in the art, including, but not limited to,nucleic acid sequencing, microarray analysis, proteomics, in-situhybridization (e.g., fluorescence in-situ hybridization (FISH)),amplification-based assays, in situ hybridization, fluorescenceactivated cell sorting (FACS), northern analysis and/or PCR analysis ofRNAs.

Nucleic Acid Detection

Nucleic acid-based methods for detection of RNA transcript expressioninclude imaging-based techniques (e.g., Northern blotting or Southernblotting), which may be used in conjunction with cells obtained from apatient following administration of, for example, a vector encoding aninterfering RNA described herein or a composition containing such aninterfering RNA construct. Northern blot analysis is a conventionaltechnique well known in the art and is described, for example, inMolecular Cloning, a Laboratory Manual, second edition, 1989, Sambrook,Fritch, Maniatis, Cold Spring Harbor Press, 10 Skyline Drive, Plainview,N.Y. 11803-2500. Typical protocols for evaluating the status of genesand gene products are found, for example in Ausubel et al., eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis).

RNA detection techniques that may be used in conjunction with thecompositions and methods described herein to evaluate the suppression ofRNA transcripts harboring expanded nucleotide repeat regions, such asDMPK RNA transcripts harboring expanded CUG trinucleotide repeats andC9ORF72 RNA transcripts harboring expanded GGGGCC (SEQ ID NO: 162)hexanucleotide repeats, further include microarray sequencingexperiments (e.g., Sanger sequencing and next-generation sequencingmethods, also known as high-throughput sequencing or deep sequencing).Exemplary next generation sequencing technologies include, withoutlimitation, Illumina sequencing, Ion Torrent sequencing, 454 sequencing,SOLiD sequencing, and nanopore sequencing platforms. Additional methodsof sequencing known in the art can also be used. For example, transgeneexpression at the mRNA level may be determined using RNA-Seq (e.g., asdescribed in Mortazavi et al., Nat. Methods 5:621-628 (2008), thedisclosure of which is incorporated herein by reference in theirentirety). RNA-Seq is a robust technology for monitoring expression bydirect sequencing the RNA molecules in a sample. Briefly, thismethodology may involve fragmentation of RNA to an average length of 200nucleotides, conversion to cDNA by random priming, and synthesis ofdouble-stranded cDNA (e.g., using the Just cDNA DoubleStranded cDNASynthesis Kit from Agilent Technology®). Then, the cDNA is convertedinto a molecular library for sequencing by addition of sequence adaptersfor each library (e.g., from Illumina®/Solexa), and the resulting 50-100nucleotide reads are mapped onto the genome.

RNA expression levels may be determined using microarray-based platforms(e.g., single-nucleotide polymorphism arrays), as microarray technologyoffers high resolution. Details of various microarray methods can befound in the literature. See, for example, U.S. Pat. No. 6,232,068 andPollack et al., Nat. Genet. 23:41-46 (1999), the disclosures of each ofwhich are incorporated herein by reference in their entirety. Usingnucleic acid microarrays, mRNA samples are reverse transcribed andlabeled to generate cDNA. The probes can then hybridize to one or morecomplementary nucleic acids arrayed and immobilized on a solid support.The array can be configured, for example, such that the sequence andposition of each member of the array is known. Hybridization of alabeled probe with a particular array member indicates that the samplefrom which the probe was derived expresses that gene. Expression levelmay be quantified according to the amount of signal detected fromhybridized probe-sample complexes. A typical microarray experimentinvolves the following steps: 1) preparation of fluorescently labeledtarget from RNA isolated from the sample, 2) hybridization of thelabeled target to the microarray, 3) washing, staining, and scanning ofthe array, 4) analysis of the scanned image and 5) generation of geneexpression profiles. One example of a microarray processor is theAffymetrix GENECHIP® system, which is commercially available andcomprises arrays fabricated by direct synthesis of oligonucleotides on aglass surface. Other systems may be used as known to one skilled in theart.

Amplification-based assays also can be used to measure the expressionlevel of a particular RNA transcript, such as a DMPK RNA transcriptharboring an expanded CUG trinucleotide repeat or a C9ORF72 RNAtranscript harboring an expanded GGGGCC (SEQ ID NO: 162) hexanucleotiderepeat. In such assays, the nucleic acid sequence of the transcript actsas a template in an amplification reaction (for example, PCR, such asqPCR). In a quantitative amplification, the amount of amplificationproduct is proportional to the amount of template in the originalsample. Comparison to appropriate controls provides a measure of theexpression level of the transcript of interest, corresponding to thespecific probe used, according to the principles described herein.Methods of real-time qPCR using TaqMan probes are well known in the art.Detailed protocols for real-time qPCR are provided, for example, inGibson et al., Genome Res. 6:995-1001 (1996), and in Heid et al., GenomeRes. 6:986-994 (1996), the disclosures of each of which are incorporatedherein by reference in their entirety. Levels of RNA transcriptexpression as described herein can be determined, for example, by RT-PCRtechnology. Probes used for PCR may be labeled with a detectable marker,such as, for example, a radioisotope, fluorescent compound,bioluminescent compound, a chemiluminescent compound, metal chelator, orenzyme.

Protein Detection

Expression of an RNA construct may also be inferred by analyzingexpression of the protein encoded by the construct. Protein levels canbe assessed using standard detection techniques known in the art.Protein expression assays suitable for use with the compositions andmethods described herein include proteomics approaches,immunohistochemical and/or western blot analysis, immunoprecipitation,molecular binding assays, ELISA, enzyme-linked immunofiltration assay(ELIFA), mass spectrometry, mass spectrometric immunoassay, andbiochemical enzymatic activity assays. In particular, proteomics methodscan be used to generate large-scale protein expression datasets inmultiplex. Proteomics methods may utilize mass spectrometry to detectand quantify polypeptides (e.g., proteins) and/or peptide microarraysutilizing capture reagents (e.g., antibodies) specific to a panel oftarget proteins to identify and measure expression levels of proteinsexpressed in a sample (e.g., a single cell sample or a multi-cellpopulation).

Exemplary peptide microarrays have a substrate-bound plurality ofpolypeptides, the binding of an oligonucleotide, a peptide, or a proteinto each of the plurality of bound polypeptides being separatelydetectable. Alternatively, the peptide microarray may include aplurality of binders, including, but not limited to, monoclonalantibodies, polyclonal antibodies, phage display binders, yeasttwo-hybrid binders, aptamers, which can specifically detect the bindingof specific oligonucleotides, peptides, or proteins. Examples of peptidearrays may be found in U.S. Pat. Nos. 6,268,210, 5,766,960, and5,143,854, the disclosures of each of which are incorporated herein byreference in their entirety.

Mass spectrometry (MS) may be used in conjunction with the methodsdescribed herein to identify and characterize transgene expression in acell from a patient (e.g., a human patient) following delivery of thetransgene. Any method of MS known in the art may be used to determine,detect, and/or measure a protein or peptide fragment of interest, e.g.,LC-MS, ESI-MS, ESI-MS/MS, MALDI-TOF-MS, MALDI-TOF/TOF-MS, tandem MS, andthe like. Mass spectrometers generally contain an ion source and optics,mass analyzer, and data processing electronics. Mass analyzers includescanning and ion-beam mass spectrometers, such as time-of-flight (TOF)and quadruple (Q), and trapping mass spectrometers, such as ion trap(IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR),may be used in the methods described herein. Details of various MSmethods can be found in the literature. See, for example, Yates et al.,Annu. Rev. Biomed. Eng. 11:49-79, 2009, the disclosure of which isincorporated herein by reference in its entirety.

Prior to MS analysis, proteins in a sample obtained from the patient canbe first digested into smaller peptides by chemical (e.g., via cyanogenbromide cleavage) or enzymatic (e.g., trypsin) digestion. Complexpeptide samples also benefit from the use of front-end separationtechniques, e.g., 2D-PAGE, HPLC, RPLC, and affinity chromatography. Thedigested, and optionally separated, sample is then ionized using an ionsource to create charged molecules for further analysis. Ionization ofthe sample may be performed, e.g., by electrospray ionization (ESI),atmospheric pressure chemical ionization (APCI), photoionization,electron ionization, fast atom bombardment (FAB)/liquid secondaryionization (LSIMS), matrix assisted laser desorption/ionization (MALDI),field ionization, field desorption, thermospray/plasmaspray ionization,and particle beam ionization. Additional information relating to thechoice of ionization method is known to those of skill in the art.

After ionization, digested peptides may then be fragmented to generatesignature MS/MS spectra. Tandem MS, also known as MS/MS, may beparticularly useful for analyzing complex mixtures. Tandem MS involvesmultiple steps of MS selection, with some form of ion fragmentationoccurring in between the stages, which may be accomplished withindividual mass spectrometer elements separated in space or using asingle mass spectrometer with the MS steps separated in time. Inspatially separated tandem MS, the elements are physically separated anddistinct, with a physical connection between the elements to maintainhigh vacuum. In temporally separated tandem MS, separation isaccomplished with ions trapped in the same place, with multipleseparation steps taking place over time. Signature MS/MS spectra maythen be compared against a peptide sequence database (e.g., SEQUEST).Post-translational modifications to peptides may also be determined, forexample, by searching spectra against a database while allowing forspecific peptide modifications.

Pharmaceutical Compositions

The interfering RNA constructs, as well as the vectors and compositionsencoding or containing these constructs, may be incorporated into avehicle for administration into a patient, such as a human patientsuffering from RNA dominance, as described herein. Pharmaceuticalcompositions containing vectors, such as viral vectors, that encode aninterfering RNA construct described herein can be prepared using methodsknown in the art. For example, such compositions can be prepared using,e.g., physiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980);incorporated herein by reference), and in a desired form, e.g., in theform of lyophilized formulations or aqueous solutions.

Mixtures of the nucleic acids and viral vectors described herein may beprepared in water suitably mixed with one or more excipients, carriers,or diluents. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms. The pharmaceuticalforms suitable for injectable use include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions (described in U.S. Pat. No.5,466,468, the disclosure of which is incorporated herein by reference).In any case the formulation may be sterile and may be fluid to theextent that easy syringability exists. Formulations may be stable underthe conditions of manufacture and storage and may be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (e.g., glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and/or vegetable oils. Proper fluidity may be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

For example, a solution containing a pharmaceutical compositiondescribed herein may be suitably buffered, if necessary, and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intrathecal, intracerebroventricular, intraparenchymal, intracisternal,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations may meet sterility, pyrogenicity, generalsafety, and purity standards as required by FDA Office of Biologicsstandards.

Routes of Administration and Dosing

Viral vectors, such as AAV vectors and others described herein,containing a transgene encoding an interfering RNA construct describedherein may be administered to a patient (e.g., a human patient) by avariety of routes of administration. The route of administration mayvary, for example, with the onset and severity of disease, and mayinclude, e.g., intravenous, intrathecal, intracerebroventricular,intraparenchymal, intracisternal, intradermal, transdermal, parenteral,intramuscular, intranasal, subcutaneous, percutaneous, intratracheal,intraperitoneal, intraarterial, intravascular, inhalation, perfusion,lavage, and oral administration. Intravascular administration includesdelivery into the vasculature of a patient. In some embodiments, theadministration is into a vessel considered to be a vein (intravenous),and in some administration, the administration is into a vesselconsidered to be an artery (intraarterial). Veins include, but are notlimited to, the internal jugular vein, a peripheral vein, a coronaryvein, a hepatic vein, the portal vein, great saphenous vein, thepulmonary vein, superior vena cava, inferior vena cava, a gastric vein,a splenic vein, inferior mesenteric vein, superior mesenteric vein,cephalic vein, and/or femoral vein. Arteries include, but are notlimited to, coronary artery, pulmonary artery, brachial artery, internalcarotid artery, aortic arch, femoral artery, peripheral artery, and/orciliary artery. It is contemplated that delivery may be through or to anarteriole or capillary.

Treatment regimens may vary, and often depend on disease severity andthe age, weight, and sex of the patient. Treatment may includeadministration of vectors (e.g., viral vectors) or other agentsdescribed herein as useful for the introduction of a transgene into atarget cell in various unit doses. Each unit dose will ordinarilycontain a predetermined-quantity of the therapeutic composition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsdescribed herein may be used, made, and evaluated, and are intended tobe purely exemplary of the invention and are not intended to limit thescope of what the inventors regard as their invention.

Example 1. Development and Evaluation of Adeno-Associated Viral VectorsEncoding miRNA Constructs for the Treatment of Disorders Associated withRNA Dominance Objective

This example describes a series of experiments conducted in order tocharacterize the development and evaluation of rAAV vectors encodingmiRNA constructs against human DMPK and murine HSA^(LR), which isexpressed in mouse models of the RNA dominance disorder, myotonicdystrophy. Myotonic dystrophy (DM) is caused by expansion of amicrosatellite repeat that leads to expression of a toxic expandedrepeat mRNA. Another RNA dominance disorder, facioscapulohumeralmuscular dystrophy (FSHD), is caused by contraction of a D4Z4macrosatellite repeat that leads to expression of DUX4, a toxic proteinin adult muscle. The goal of the experiments described in this exampleis to develop AAV vectors encoding miRNA constructs that target muscleDM and FSHD-related mRNAs, so as to prevent muscle dysfunction and lossin individuals with disease.

Materials and Methods

To develop rAAV-RNAi therapy for myotonic dystrophy type 1 (DM1), ahuman skeletal actin gene (HSA) directed interfering RNA hairpin wasdesigned and produced so that its efficacy could be tested in theHSA^(LR) mouse model of DM1. The HSA^(LR) mouse was produced withinsertion of an expanded CTG repeat in the 3′ UTR of the HSA gene, asimilar genetic context to the disease-causing repeat expansion in theDMPK gene in humans. The HSA^(LR) mouse displays many of the genetic andphenotypic changes associated with DM1 in skeletal muscle, includingmyotonia, splicing changes in a variety of mRNAs, and nuclear inclusions(foci). Among the aims of this study was to translate the approach ofreducing expression of aberrant HSA^(LR) RNA transcripts containingexpanded CUG repeat regions to develop a paradigm for suppressing thehuman disease target gene, DMPK.

To this end, RNAi expression cassettes with 19-22 bp target recognitionsequences were tested for a variety of applications. The RNAi hairpinswere based on miR30a endogenous sequence. The single-stranded rAAVgenomes were packaged in an rAAV6 capsid for targeting muscle. rAAV6 HSARNAi constructs were delivered by intravenous injection (IV) previouslyinto the tail vein of HSA^(LR) mice at 4 weeks of age (n=7-9).

Results

As shown in FIGS. 2A-2C, three generations of rAAV-RNAi vectors weredeveloped to target several genes for testing and development oftherapeutic RNAi. The rAAV plasmid pARAP4 includes the human alkalinephosphatase reporter gene (Hu Alk Phos) reporter gene, expressed fromthe Rous Sarcoma Virus (RSV) promoter, and the SV40 polyadenylationsequence, pA. The inverted terminal repeats (ITRs) originate from rAAV2and genomes are packaged in rAAV6 capsids. The newest vectormodification removes the RSV promoter sequence to prevent Hu Alk Phosexpression that limited rAAV-RNAi efficacy at higher doses due musclecell toxicity.

As show in FIGS. 3A-3C, HSA^(LR) mice show characteristics of musculardystrophy resembling DM in humans. The HSA^(LR) transgene is derivedfrom insertion of a (CTG)₂₅₀ repeat in the 3′ UTR of the HSA gene. Whenthe transgene is expressed in mouse skeletal muscle, myotonic dischargesare evident, splicing alterations occur in a variety of mRNAs, andnuclear foci containing the expanded transgenic mRNA and splicingfactors are present.

As shown in FIG. 4, HSA^(LR) mice were transduced with rAAV6 HSA miRDM10. Human placental alkaline phosphatase (AP) staining indicatespresence of the viral genome with active reporter gene expression, andH&E staining of cryosections from treated mice is also shown. Timepoint,8 weeks post-injection, 4 week-old HSA^(LR) mice.

As shown in FIGS. 5A and 5B, FIGS. 6A-6E, and FIGS. 7A-7C, rAAV-RNAivectors encoding miRNA constructs complimentary to HSA^(LR) transcriptsreduced the expression of pathologic HSA^(LR) RNA and effectuated therelease of RNA-binding splicing factors that would otherwise besequestered by the CUG repeat expansion, as evidenced by the restorationof correct splicing of SERCA1 mRNA (FIGS. 6C and 6D). rAAV6 HSA miR DM10systemic injection improves splicing of SERCA1 and CLCN1 in the tibialisanterior (TA) muscle. In contrast, a different RNAi hairpin, miR DM4,was not as effective at reversing these splicing defects.

Selection of DMPK Regions for RNAi

Selection of DMPK RNA target sequences for incorporation intomiRNA-based hairpins was done using guidelines for siRNA design.Candidate target sequences were eliminated based on predicted seedsequence matches at other loci or in alternatively spliced regions.Additional screening included analysis using Bowtie for short sequencealignment to the human genome and extensive BLAST searches. Exemplaryinterfering RNA constructs against human DMPK are shown in Table 4,above, and are represented graphically in FIG. 1.

Conclusions

As demonstrated in these experiments, local injection of vectors lackingthe Hu Alk Phos promoter in muscle improves the splicing-relatedphenotype of the HSA^(LR) mice, as measured by the increase in amount ofSERCA1 mRNA adult splicing product. Splicing for SERCA1 mRNA wasapproximately 95% corrected and demonstrates that improvements intechnology have the potential to increase the efficacy of this approach.

Additionally, these results demonstrate that rAAV6-mediated delivery ofthe HSA hairpins improves many molecular and phenotypic characteristicsof DM1 modeled in the HSA^(LR) mouse, including myotonia,disease-related splicing changes, and sequestration of splicing factors.Moreover, using the compositions and methods described herein, vectorscarrying U6-expressed DMPK miRNAs can reduce endogenous DMPK mRNA inHEK293 cells following transfection. These data indicate the utility foran RNAi-mediated gene therapy to treat DM1.

rAAV-mediated therapy is effective for spinal muscular atrophy and isprogressing in clinical trials for Duchenne muscular dystrophy. Studiesin non-human primates demonstrate that AAV can transduce muscleefficiently with regional limb delivery and persist for as long as 10years. These studies support development of rAAV-mediated RNAi genetherapy for the treatment of dominant muscle diseases in humans, such asmyotonic dystrophy type I, among others described herein.

Example 2. Treatment of Myotonic Dystrophy in a Human Patient byAdministration of a Viral Vector Encoding a miRNA Against DMPK

Using the compositions and methods described herein, a physician ofskill in the art may administer to a patient having myotonic dystrophytype I a viral vector encoding a miRNA that anneals to, and reduces theexpression of, mutant DMPK RNA transcripts containing expanded CUGrepeat regions. The vector may be an AAV vector, such as a pseudotypedAAV2/8 or AAV2/9 vector. The vector may be administered by way of one ormore routes of administration described herein, such as by intravenous,intrathecal, intracerebroventricular, intraparenchymal, intracisternal,intramuscular, or subcutaneous injection. The encoded miRNA may be amiRNA characterized herein, such as a miRNA having the nucleic acidsequence of any one of SEQ ID Nos: 40-161.

Following administration of the vector, the physician may monitor theprogression of the disorder and the efficacy of the treatment byassessing, for example, the concentration of correctly spliced mRNAtranscripts encoding SERCA1, CLCN1, and/or ZASP, using an RNA detectiontechnique described herein. The physician may also monitor theconcentration of functional SERCA1, CLCN1, and/or ZASP protein productresulting from the correctly spliced transcripts. Additionally oralternatively, the physician may monitor the concentration of mutantDMPK RNA transcripts expressed by the patient, particularly, DMPKtranscripts having from about 50 to about 4,000, or more, CUGtrinucleotide repeats. A finding that (i) the concentration of correctlyspliced SERCA1, CLCN1, and/or ZASP mRNA transcripts has increased, (ii)the concentration of functional SERCA1, CLCN1, and/or ZASP proteinproducts resulting from translation of the correctly spliced mRNAtranscripts has increased, and/or (iii) the concentration of mutant DMPKRNA transcripts harboring expanded CUG trinucleotide repeat regions hasdecrease may serve as an indicator that the patient has beensuccessfully treated. The physician may also monitor the progression ofone or more symptoms of the disease, such as myotonia, muscle stiffness,disabling distal weakness, weakness in the face and jaw muscles,difficulty in swallowing, drooping of the eyelids (ptosis), weakness ofneck muscles, weakness in arm and leg muscles, persistent muscle pain,hypersomnia, muscle wasting, dysphagia, respiratory insufficiency,irregular heartbeat, heart muscle damage, apathy, insulin resistance,and cataracts. In children, symptoms may also include developmentaldelays, learning problems, language and speech difficulties, andpersonality development challenges. A finding that one or more, or all,of the foregoing symptoms has been ameliorated may also serve as aclinical indicator of successful treatment.

Example 3. Treatment of Myotonic Dystrophy in a Human Patient byAdministration of an siRNA Oligonucleotide Against DMPK

Using the compositions and methods described herein, a physician ofskill in the art may administer to a patient having myotonic dystrophytype I an siRNA oligonucleotide that anneals to, and reduces theexpression of, mutant DMPK RNA transcripts containing expanded CUGrepeat regions. The oligonucleotide may have, for example, the nucleicacid sequence of any one of SEQ ID NOs: 3-39.

Following administration of the oligonucleotide, the physician maymonitor the progression of the disorder and the efficacy of thetreatment by assessing, for example, the concentration of correctlyspliced mRNA transcripts encoding SERCA1, CLCN1, and/or ZASP, using anRNA detection technique described herein. The physician may also monitorthe concentration of functional SERCA1, CLCN1, and/or ZASP proteinproduct resulting from the correctly spliced transcripts. Additionallyor alternatively, the physician may monitor the concentration of mutantDMPK RNA transcripts expressed by the patient, particularly, DMPKtranscripts having from about 50 to about 4,000, or more, CUGtrinucleotide repeats. A finding that (i) the concentration of correctlyspliced SERCA1, CLCN1, and/or ZASP mRNA transcripts has increased, (ii)the concentration of functional SERCA1, CLCN1, and/or ZASP proteinproducts resulting from translation of the correctly spliced mRNAtranscripts has increased, and/or (iii) the concentration of mutant DMPKRNA transcripts harboring expanded CUG trinucleotide repeat regions hasdecrease may serve as an indicator that the patient has beensuccessfully treated. The physician may also monitor the progression ofone or more symptoms of the disease, such as myotonia, muscle stiffness,disabling distal weakness, weakness in the face and jaw muscles,difficulty in swallowing, drooping of the eyelids (ptosis), weakness ofneck muscles, weakness in arm and leg muscles, persistent muscle pain,hypersomnia, muscle wasting, dysphagia, respiratory insufficiency,irregular heartbeat, heart muscle damage, apathy, insulin resistance,and cataracts. In children, symptoms may also include developmentaldelays, learning problems, language and speech difficulties, andpersonality development challenges. A finding that one or more, or all,of the foregoing symptoms has been ameliorated may also serve as aclinical indicator of successful treatment.

Example 4. Ability of Anti-DMPK siRNA Molecules to Suppress DMPK1Expression in Cultured HEK293 Cells

This example describes the results of experiments conducted in order toevaluate the ability of anti-DMPK siRNA molecules, such as various siRNAmolecules described in Table 4 herein, to attenuate the expression ofDMPK1 mRNA in cultured human cells. For purposes of comparison, inaddition to testing siRNA molecules described in Table 4, above, ascrambled siRNA molecule and commercially available anti-DMPK siRNAmolecules were tested as well.

To conduct these experiments, HEK293 cells (2×10⁵ cells/well) weretransfected in triplicate with either 5 μM of a candidate anti-DMPKsiRNA molecule (such as an siRNA molecule described in Table 4, above)or 5 μM scrambled negative siRNA control (Silencer® Select siRNA, Ambionby Life Technologies) using 1 μL Lipofectamine™ RNAiMAX (Thermo FisherScientific). Mock transfections of cells treated only with 1 μLLipofectamine™ RNAiMAX were included for normalization. RNA washarvested after 48 hours using the RNeasy Plus Mini Kit (Qiagen). cDNAgenerated was subsequently generated using SuperScript™ III ReverseTranscriptase (Thermo Fisher Scientific) using 150 ng of RNA per sample.qPCR was performed to detect DMPK1 knockdown. qPCR experiments were setup in triplicate using the TaqMan™ Fast Advanced Master Mix, andreactions were performed using a QuantStudio 3 RT-PCR instrument (ThermoFisher Scientific). DMPK1 expression values were normalized to GAPDH(TaqMan™ Gene expression assay ID Hs02786624_g1) using QuantStudio 3software.

The results of these experiments are shown in FIG. 9. As evidenced bythis figure, various siRNA molecules described in Table 4, above, arecapable of downregulating DMPK1 expression in live human cells. The datashown graphically in FIG. 9 are provided in numerical form in Table 6,below.

TABLE 6 Suppression of DMPK1 expression in HEK293 cells by siRNAmolecules in FIG. 9 siRNA Molecule Normalized DMPK1 Expression SEQ IDNO: 19 36.535% SEQ ID NO: 6 45.575% SEQ ID NO: 20 45.748% Anti-DMPKn351357 51.946% SEQ ID NO: 5 60.258% Anti-DMPK HSS1028253 60.498% SEQ IDNO: 4 60.926% Anti-DMPK HSS176211 63.934% SEQ ID NO: 10 64.104% SEQ IDNO: 16 66.067% SEQ ID NO: 23 68.659% SEQ ID NO: 24 79.758% SEQ ID NO: 780.929% SEQ ID NO: 22 84.449% SEQ ID NO: 21 85.495% Anti-DMPK n35135886.927% Anti-DMPK s4165 93.519% SEQ ID NO: 18 97.217% SEQ ID NO: 17122.154%

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from theinvention that come within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

What is claimed is:
 1. A viral vector comprising one or more transgenes,each encoding an interfering ribonucleic acid (RNA) of at least 17nucleotides in length, wherein each interfering RNA comprises a portionthat anneals to an endogenous RNA transcript comprising an expandedrepeat region, and wherein the portion of each interfering RNA annealsto a segment of the endogenous RNA transcript that does not overlap withthe expanded repeat region.
 2. The viral vector of claim 1, wherein theendogenous RNA transcript encodes human dystrophia myotonica proteinkinase (DMPK).
 3. The viral vector of claim 2, wherein the expandedrepeat region comprises 50 or more CUG trinucleotide repeats. 4.(canceled)
 5. The viral vector of claim 2, wherein the vector furthercomprises a transgene encoding a human DMPK RNA transcript that does notanneal to the interfering RNA.
 6. The viral vector of claim 5, whereinthe human DMPK RNA transcript is less than 85% complementary to theinterfering RNA.
 7. The viral vector of claim 2, wherein the endogenousRNA transcript comprises a portion having at least 85% sequence identityto the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO:
 2. 8-11.(canceled)
 12. The viral vector of claim 2, wherein the portion of eachinterfering RNA has a nucleic acid sequence that is least 85%complementary to the nucleic acid sequence of a segment within any oneof exons 1-15 of human DMPK, any one of introns 1-14 of human DMPK, anexon-intron boundary within human DMPK, or within the 5′ untranslatedregion (UTR) or 3′ UTR of human DMPK. 13-33. (canceled)
 34. The viralvector of claim 2, wherein the portion of each interfering RNA annealsto a segment of the endogenous RNA transcript having the nucleic acidsequence of any one of SEQ ID NOs: 3-39. 35-38. (canceled)
 39. The viralvector of claim 2, wherein the interfering RNA comprises a portionhaving at least 85% sequence identity to the nucleic acid sequence ofany one of SEQ ID NOs: 3-161. 40-42. (canceled)
 43. The viral vector ofclaim 1, wherein the endogenous RNA transcript comprises humanchromosome 9 open reading frame 72 (C9ORF72) and an expanded repeatregion. 44-49. (canceled)
 50. The viral vector of claim 43, wherein theportion of each interfering RNA has a nucleic acid sequence that is atleast 85% complementary to the nucleic acid sequence of a segment withinhuman C9ORF72. 51-60. (canceled)
 61. The viral vector of claim 1,wherein the interfering RNA is a short interfering RNA (siRNA), a shorthairpin RNA (shRNA), or a micro RNA (miRNA). 62-64. (canceled)
 65. Theviral vector of claim 1, wherein the interfering RNA is operably linkedto a promoter that induces expression of the interfering RNA in a musclecell or neuron.
 66. The viral vector of claim 65, wherein the promoteris a desmin promoter, a phosphoglycerate kinase (PGK) promoter, a musclecreatine kinase promoter, a myosin light chain promoter, a myosin heavychain promoter, a cardiac troponin C promoter, a troponin I promoter, amyoD gene family promoter, an actin alpha promoter, an actin betapromoter, an actin gamma promoter, or a promoter within intron 1 ofocular paired like homeodomain 3 (PITX3).
 67. (canceled)
 68. The viralvector of claim 1, wherein the viral vector is an adeno-associated virus(AAV), optionally wherein the AAV is an AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74 serotype.
 69. (canceled) 70.The viral vector of claim 68, wherein the viral vector is AAV2/8 orAAV2/9. 71-74. (canceled)
 75. A nucleic acid encoding or comprising aninterfering RNA, wherein the interfering RNA comprises a portion havingat least 85% sequence identity to the nucleic acid sequence of any oneof SEQ ID NOs: 3-161. 76-116. (canceled)
 117. A method of reducing theoccurrence of spliceopathy in a human patient in need thereof, themethod comprising administering to the patient a therapeuticallyeffective amount of the vector of claim
 1. 118. The method of claim 117,wherein the patient has myotonic dystrophy or amyotrophic lateralsclerosis. 119-124. (canceled)
 125. A method of treating a disordercharacterized by nuclear retention of RNA comprising an expanded repeatregion in a human patient in need thereof, the method comprisingadministering to the patient a therapeutically effective amount of thevector of claim
 1. 126-130. (canceled)